3-dimensional cardiac fibroblast derived extracellular matrix

ABSTRACT

A bioscaffold made from an isolated cardiac fibroblast-derived 3-dimensional extracellular matrix (ECM) is disclosed. The bioscaffold can be used as an epicardial patch for the delivery of therapeutic cells into myocardial tissue. Methods of making the 3-dimensional extracellular matrix using cultured cardiac fibroblasts are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/094,619, filed Apr. 8, 2016, which is a divisional of U.S. patentapplication Ser. No. 14/330,486, filed Jul. 14, 2014, now U.S. Pat. No.9,744,265, which is a divisional of U.S. patent application Ser. No.13/593,969, filed Aug. 24, 2012, now U.S. Pat. No. 8,802,144, whichclaims the benefit of U.S. provisional Application No. 61/575,658 filedon Aug. 25, 2011. Each of these applications is incorporated byreference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH/DEVELOPMENT

This invention was made with government support under HL092477 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

The human heart continuously undergoes slow cellular turnover throughapoptosis of cardiomyocytes and proliferation of cardiac progenitorcells at a rate of approximately 1% turnover a year (see Bergmann, O.,et al., Evidence for cardiomyocyte renewal in humans. Science, 2009.324(5923): p. 98-102). This slow cardiomyocyte turnover is important formaintaining cardiac function from birth to old age; however, residentcardiac progenitor cells have shown little capacity for robust cardiacregeneration following myocardial injury such as that caused by amyocardial infarction (see Bolli, P. and H. W. Chaudhry, Molecularphysiology of cardiac regeneration. Ann N Y Acad Sci. 1211: p. 113-26).Thus, it is likely that any successful post-injury progenitor cell-basedcardiac regeneration strategy would require administering progenitorcells such as stem cells from a source outside of the native injuredcardiac tissue.

A major obstacle in developing cell-based regenerative strategies is theneed to successfully transfer a sufficient number of therapeutic cellsto the target location (Karp, J. M. and G. S. Leng Teo, Mesenchymal stemcell homing: the devil is in the details. Cell Stem Cell, 2009. 4(3): p.206-16). For example, in the infarcted heart, transfer of therapeuticcells is challenged not only by the motion of the heart but also by itsheightened electrical and structural instability (Fernandes, S., et al.,Autologous myoblast transplantation after myocardial infarctionincreases the inducibility of ventricular arrhythmias. Cardiovasc Res,2006. 69(2): p. 348-58). Despite these difficulties, some reportsindicate that intramyocardial or intracoronary injection of potentiallytherapeutic cells following cardiac injury can result in modestimprovement in cardiac function (see, e.g., Bolli, R., et al., Cardiacstem cells in patients with ischaemic cardiomyopathy (SCIPIO): initialresults of a randomised phase 1 trial. Lancet, 2011. 378(9806): p.1847-57; Price, M. J., et al., Intravenous mesenchymal stem cell therapyearly after reperfused acute myocardial infarction improves leftventricular function and alters electrophysiologic properties. Int JCardiol, 2006. 111(2): p. 231-9). However, to date, no study hasreported the large scale engraftment of cells or functionalcardiomyocyte regeneration in an infarcted heart.

An alternative to direct injection of potentially therapeutic cells isto place them in a biomatrix “patch” that can be affixed to a desiredarea of the heart, such as onto the epicardium. In this way, a patchpre-seeded with potentially therapeutic cells may release the cells,allowing them to migrate into the injured myocardium to facilitateregeneration directly, or may retain the cells in close proximity to theinjury to facilitate regeneration indirectly (i.e., via paracrinefactors) (see Gnecchi, M., et al., Paracrine mechanisms in adult stemcell signaling and therapy. Circ Res, 2008. 103(11): p. 1204-19). Thisapproach has been attempted using patches made of synthetic materials(see Silva, E. A. and D. J. Mooney, Synthetic extracellular matrices fortissue engineering and regeneration. Curr Top Dev Biol, 2004. 64: p.181-205), as well as naturally occurring biomaterials such as theextracellular matrix (ECM) remaining after decellularization of heartvalves (Bader, A., et al., Tissue engineering of heart valves—humanendothelial cell seeding of detergent acellularized porcine valves. EurJ Cardiothorac Surg, 1998. 14(3): p. 279-84), skeletal muscle (Borschel,G. H., R. G. Dennis, and W. M. Kuzon, Jr., Contractile skeletal muscletissue-engineered on an acellular scaffold. Plast Reconstr Surg, 2004.113(2): p. 595-602; discussion 603-4), or bovine dermis (Kouris, N. A.,et al., Directed Fusion of Mesenchymal Stem Cells with Cardiomyocytesvia VSV-G Facilitates Stem Cell Programming. Stem Cells Int, 2012. 2012:p. 414038). Notably, none of these patches are made from myocardialtissue, nor do any of them exhibit the unique structural characteristicsof cardiac ECM.

The extracellular matrix (ECM) is the extracellular part of animaltissue that provides structural support to the cells, in addition toperforming various other important functions. As such, it is thedefining feature of connective tissue in animals. Fibroblasts play acentral role in the synthesis and maintenance of the ECM. In vivo, theECM has a 3-dimensional structure, which facilitates interaction on allsides of the cells that are associated with the ECM. Specifically, cellsassociated with the ECM may be in contact with ECM surfaces both aboveand below the cells. To accurately model ECM-cell interactions in vivo,any ECM structure that is used in vitro would likewise need to have atrue 3-dimensional structure. A 3-dimensional ECM cannot besubstantially flat, and would have a thickness of at least 20 μm. Thecardiac ECM is a unique 3-dimensional structure that facilitates thenormal functioning of the heart. The arrangement of the cardiac ECMhelps channel the contraction of each myocyte into one forcefulcontraction, ultimately ejecting blood from the ventricles into thecirculation (see Akhyari, P., et al., Myocardial tissue engineering: theextracellular matrix. Eur J Cardiothorac Surg, 2008. 34(2): p. 229-41).Furthermore, the cardiac ECM has importance beyond providing structureto cardiac tissue. Specifically, it plays a role in cardiac woundhealing (see Dobaczewski, M., et al., Extracellular matrix remodeling incanine and mouse myocardial infarcts. Cell Tissue Res, 2006. 324(3): p.475-88; Jourdan-Lesaux, C., J. Zhang, and M. L. Lindsey, Extracellularmatrix roles during cardiac repair. Life Sci. 87(13-14): p. 391-400),and may play a role in cardiac regeneration (see Akhyari, P., et al.,Myocardial tissue engineering: the extracellular matrix. Eur JCardiothorac Surg, 2008. 34(2): p. 229-41). Although the production of athin (<0.1 μm), 2-dimensional putative cardiac ECM on the surface ofculture plate has been previously reported (see VanWinkle, W. B., M. B.Snuggs, and L. M. Buja, Cardiogel: a biosynthetic extracellular matrixfor cardiomyocyte culture. In Vitro Cell Dev Biol Anim, 1996. 32(8): p.478-85), to our knowledge, there has been no previous report of the invitro production of a 3-dimensional cardiac ECM.

It is increasingly recognized that ECM is highly tissue-specific, withthe fibroblasts of a given tissue synthesizing an ECM having a uniquecombination of structural proteins and bioactive molecules (i.e. growthfactors). Accordingly, transplantation of cells across different tissuetypes could be problematic (see Badylak, S. F., D. O. Freytes, and T. W.Gilbert, Extracellular matrix as a biological scaffold material:Structure and function. Acta Biomater, 2009. 5(1): p. 1-13).

In addition to not being of myocardial origin, biomaterials currentlyunder investigation for use in cardiac cell transfer patches have othernotable limitations. For example, a frequent issue with the use ofsynthetic or decellularized tissues in patches for therapeutic celldelivery to the myocardium is the inability of the patch to physicallyadhere to the epicardial surface. The patches often require the use ofglue or sutures to hold the patch to the heart (see, e.g., Fiumana, E.,et al., Localization of mesenchymal stem cells grafted with ahyaluronan-based scaffold in the infarcted heart. J Surg Res, 2012). Ifthe patch does not maintain firm contact with the surface of the heart,the ability of the cells to transfer is decreased significantly.Furthermore, epicardial patches must not only adhere to the heart, butmust also have the proper tensile strength and compliance to toleratecardiac movement. If a patch's compliance does not match that of theventricle and does not move with the beating heart, gaps may form underthe surface, reducing cell transfer. Epicardial patches lacking tensilestrength may disintegrate under the strains of a beating heart.

For these reasons, a patch made from a 3-dimensional bioscaffold that iscardiac-specific would be highly desirable to facilitate successfuldelivery of therapeutic cells to injured or diseased myocardial tissue.

BRIEF SUMMARY OF THE INVENTION

This application discloses an epicardial patch for facilitating thedelivery of cells to myocardial tissue, as well the isolated cardiacextracellular matrix that makes up the patch and methods of making andusing the same. Advantageously, the isolated cardiac extracellularmatrix is truly 3-dimensional, is myocardial in origin and composition,adheres well to the wall of the heart without the need for glue orsutures, moves flexibly with the heart, and successfully facilitates thedelivery of seeded cells into myocardial tissue.

In a first aspect, the disclosure encompasses a bioscaffold forfacilitating the delivery of cells to myocardial tissue. The bioscaffoldis made up of an isolated 3-dimensional cardiac extracellular matrix(ECM) derived from cardiac fibroblast cells in vitro. The isolatedcardiac ECM has a composition similar to the in vivo 3-dimensionalextracellular matrix that is unique to cardiac tissue. Along with othercomponents, it is made up largely of the structural proteinsfibronectin, collagen type I, collagen type III, and elastin, and thebioscaffold has a thickness of 20-500 μm. In some embodiments, thebioscaffold has a thickness range of 30-200 μm or of 50-150 μm. In someembodiments, fibronectin molecules make up from 60% to 90% of thestructural protein molecules present in the ECM. In some embodiments,fibronectin molecules make up from 70% to 90% of the structural proteinmolecules present in the ECM. In some embodiments, fibronectin moleculesmake up from 80% to 90% of the structural protein molecules present inthe ECM.

In some embodiments, the ECM further includes the structural proteincollagen type V. Other structural proteins may also be included in thecardiac ECM. Preferably, the structural proteins of the cardiac ECM arenot chemically cross-linked.

In addition to the structural proteins, the cardiac ECM may includematricellular proteins, such as growth factors and cytokines, as well asother substances. Non-limiting examples of other proteins that may befound in the cardiac ECM include latent transforming growth factor beta1 (LTGFB-1), latent transforming growth factor beta 2 (LTGFB-2),connective tissue growth factor (CTGF), secreted protein acidic and richin cysteine (SPARC), versican core protein (VCAN), galectin 1, galectin3, matrix gla protein (MGP), sulfated glycoprotein 1, protein-lysine6-oxidase, and biglycan.

In certain preferred embodiments, the cardiac ECM is not attached to asolid surface, so that the bioscaffold can be readily used as anepicardial patch that can be applied to the heart wall. Optionally, thecardiac ECM is decellularized, and is thus essentially devoid of intactcardiac fibroblast cells. In some embodiments, the bioscaffold may beseeded with one or more cells that are therapeutic for cardiac diseaseor injury. Examples of therapeutic cells types that could be used toseed the bioscaffold include without limitation skeletal myoblasts,embryonic stem cells (ES), induced pluripotent stem cells (iPS),multipotent adult germline stem cells (maGCSs), bone marrow mesenchymalstem cells (BMSCs), very small embryonic-like stem cells (VSEL cells),endothelial progenitor cells (EPCs), cardiopoietic cells (CPCs),cardiosphere-derived cells (CDCs), multipotent Is/1+ cardiovascularprogenitor cells (MICPs), epicardium-derived progenitor cells (EPDCs),adipose-derived stem cells, human mesenchymal stem cells (derived fromiPS or ES cells), human mesenchymal stromal cells (derived from iPS orES cells) skeletal myoblasts, or combinations thereof. Such embodimentsalso encompass methods for treating a subject having a cardiac diseaseor injury, wherein the surface of the subject's heart is contacted witha seeded epicardial patch as described above, and wherein the severityof the cardiac disease or injury is decreased. The method can begenerally used in the treatment of any cardiac disease or injury whereincardiomyocytes are lost, including without limitation in treating injurycaused by an acute myocardial infarct, by a bacterial infection, by aviral infection, by congenital defect, or by heart failure.

In a second aspect, the disclosure encompasses a method for preparing a3-dimensional cardiac extracellular matrix. The method includes thesteps of (a) isolating cardiac fibroblasts from cardiac tissue; (b)expanding the cardiac fibroblasts in culture for 1-7 passages; and (c)plating the expanded cardiac fibroblasts into a culture having a celldensity of 100,000 to 500,000 cells per cm². Optionally, step (c)involves plating the expanded cardiac fibroblasts into a culture havinga cell density of 100,000 to 200,000 cells per cm². Under theseconditions, the cardiac fibroblasts secrete a 3-dimensional cardiacextracellular matrix having a thickness of 20-500 μm that is attached tothe surface on which the expanded cardiac fibroblasts are plated.Optionally, the cardiac ECM may have a thickness of 30-200 μm or 50-150μm.

Optionally, the secreted cardiac ECM may be contacted withethylenediaminetetraaceticacid (EDTA) to detach the cardiac ECM from thesurface on which it is plated. The detached cardiac ECM forms a freefloating bioscaffold that can be seeded with therapeutic cells and usedas an epicardial patch, as described previously.

The cardiac ECM formed in the method can optionally be decellularized toremove any remaining cardiac fibroblasts from the ECM. Anydecellularizing agent may be used to decellularize the cardiac ECM.Non-limiting examples of decellurizing agents known in the art includeenzymatic agents, such as trypsin, endonucleases, or exonucleases;chemical agents, such as alkaline or acid solutions, hypertonic orhypertonic solutions, ethylenediaminetetraacetic acid (EDTA), ethyleneglycol tetraacetic acid (EGTA), ammonium hydroxide, and peracetic acid;nonionic detergents, such as octylphenol ethylene oxide (Triton™-X 100);ionic detergents, such as sodium dodecyl sulfate (SDS) and polyethersulfonate (Triton™-X 200); and zwitterionic detergents, such as3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),sulfobetaine-10 and -16, and tri(n-butyl)phosphate.

In one embodiment, decellularization is performed by contacting thecardiac ECM with peracetic acid and subsequently rinsing the cardiacextracellular matrix with water. Alternatively, decellularization isperformed by contacting the secreted cardiac extracellular matrix withammonium hydroxide and octylphenol ethylene oxide (Triton™ X-100) andsubsequently rinsing the cardiac extracellular matrix with water.

Optionally, the method further includes the step of seeding the cardiacextracellular matrix with one or more cells that are therapeutic forcardiac disease or injury. Examples of such therapeutic cells includewithout limitation skeletal myoblasts, embryonic stem cells (ES),induced pluripotent stem cells (iPS), multipotent adult germline stemcells (maGCSs), bone marrow mesenchymal stem cells (BMSCs), very smallembryonic-like stem cells (VSEL cells), endothelial progenitor cells(EPCs), cardiopoietic cells (CPCs), cardiosphere-derived cells (CDCs),multipotent Is/1+ cardiovascular progenitor cells (MICPs),epicardium-derived progenitor cells (EPDCs), adipose-derived stem cells,human mesenchymal stem cells (derived from iPS or ES cells), humanmesenchymal stromal cells (derived from iPS or ES cells) skeletalmyoblasts, or combinations thereof.

This aspect of the disclosure further encompasses a bioscaffold forfacilitating cell delivery to myocardial tissue (i.e. an epicardialpatch) that is made from the isolated 3-dimensional cardiac ECM producedby the method described above.

The disclosed compositions and methods are further detailed below.

BRIEF DESCRIPTION OF DRAWINGS

The invention will be better understood and features, aspects, andadvantages other than those set forth above will become apparent whenconsideration is given to the following detailed description thereof.Such detailed description makes reference to the following drawings.

FIGS. 1 A and B are photographs of representative paraffin embeddedimmunostained CF 3D-ECM decellularized with AH buffer. A) and B) stainedfor fibronectin, collagen type 1, and DAPI. Individual colors not shown.Scale bar=10 μm.

FIG. 2 is a photograph showing a cardiac fibroblast patch in a 40 mmdiameter culture dish.

FIG. 3 are photographs showing BMSC surface marker expression. A) CD29B) CD44 C) c-Kit.

FIG. 4 are photographs showing BMSC morphology. A) BMSC grown on glasscoverslips, stained for CD29, CD44, and c-kit (colors not shown). B)BMSC grown on CF 3D-ECM, stained for CD29, collagen type 1, CD44 (colorsnot shown). Scale bar=10 μm.

FIG. 5 is a bar graph showing that culturing BMSC on CF 3D-ECM increasesproliferation of the cells, as compared to culturing the cells on tissueculture plastic. BMSC cultured on AH decellularized matrix hadsignificantly increased proliferation compared to BMSC cultured on PAAdecellularized matrix (n=5-6/group).

FIG. 6 is a schematic diagram showing the experimental design of geneanalysis of BMSC cultured on AH, PAA, or P1 (cultured on tissueculture-treated plastic (TCP)).

FIG. 7 shows the oscillatory flow device used in the reportedexperiments. A) Side images of passive pumping via dispensed droplets.B) Top view of microchannel and diaphragm assembly. Inset shows 7×1array of microchannels on a single microscope slide. C) Side view ofsetup. Cantilever piezoelectric actuator deflects diaphragm according toapplied voltage signals. At low frequency and moderate amplitude, thiscreates a volume change in the air cavity and displaces fluid in thechannel.

FIG. 8 shows the placement of epicardial patches. A) Representativeheart before patch placement. B) A CF 3D-ECM patch placed (arrow). C) ACF patch (arrow), the suture used to occlude the coronary artery isvisible under the patch.

FIG. 9 shows representative examples of control hearts (A), hearts withCF 3D-ECM patches (B,C) and hearts with CF patches (D,E). Arrowsindicate patches.

FIG. 10 shows H&E staining of representative hearts (dark spots=nuclei).A) 4× control heart, B) 10× control heart, C) 4× heart with CF 3D-ECMpatch, D) 10× heart with CF 3D-ECM patch, E) 4× heart with CF patch(arrows), F) 20× heart with CF patch; arrows depict interface of patchand heart.

FIG. 11 shows A) H&E staining of CF 3D-ECM patch (left), area in the boxis stained for fibronectin and CD146 (right), arrows denote theinterface of the patch and heart. B) H&E staining of CF patch (right),area in the box is stained for fibronectin and CD146 (left), arrowsdenote the interface of the patch and heart.

FIG. 12 is a representative example of hMSC CD73 expression in the CF3D-ECM.

FIG. 13 is a bar graph showing the protein composition of the CF-ECMpatch. Note the high fibronectin content.

FIG. 14 shows photographs of CF-ECM patch. A) Shows surface staining forCF-ECM patch. B) Shows cross section of CF-ECM patch, stained forfibronectin, collagen type I, and DAPI (colors not shown) (scale bar=50μm). Note the thickness of approximately 150 μm. C) Shows scanningelectron micrograph of CF-ECM surface (scale bar=40 μm).

FIG. 15 shows photographs of CF-ECM patch. A) Shows CF-ECM at time ofplacement on the mouse heart (0 h), arrows denote the edge of the patch.B) Shows attached patch after 48 h on the beating mouse heart, arrowsdenote the edge of the patch. C) Shows hematoxylin and eosin stain of across-section of the epicardial surface, arrows denote the patch. Notethe absence of gaps between the patch and epicardial surface (scalebar=100 μm). D) Shows immunofluorescence micrograph of an attached patchafter 48 hours on the beating mouse heart (scale bar=100 μm). Insetimage denotes the tight attachment between CF-ECM patch and theepicardial surface. Stained for Fibronectin and DAPI (colors not shown)(scale bar=25 μm).

FIG. 16 shows photographs of FISH staining for human centromeres (brightspots). Nuclei indicates the presence of human cells within the mouseheart. Note that cardiomyocytes are highly autofluorescent helping todistinguish hMSCs from myocytes. A) The epicardial surface with attachedpatch, note the presence of hMSCs in the CF-ECM patch. The dashed lineindicates the interface between the CF-ECM patch and the epicardium(scale bar=50 μm). B) Mid-myocardium with human nuclei (scale bar=50μm). C) Endocardial surface with human nuclei present (scale bar=10 μm).

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and are herein described in detail. It shouldbe understood, however, that the description herein of specificembodiments is not intended to limit the invention to the particularforms disclosed, but on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

This application discloses an epicardial patch for facilitating thedelivery of cells to myocardial tissue, as well as the isolated cardiacextracellular matrix that makes up the patch and methods of making andusing the same. Advantageously, the isolated cardiac extracellularmatrix is truly 3-dimensional, is myocardial in origin and composition,adheres well to the wall of the heart without the need for glue orsutures, moves flexibly with the heart, and successfully facilitates thedelivery of seeded cells to myocardial tissue.

In a first aspect, the disclosure encompasses a bioscaffold forfacilitating the delivery of cells to myocardial tissue. The bioscaffoldis made up of an isolated 3-dimensional cardiac extracellular matrix(ECM) derived from cardiac fibroblast cells in vitro. By “isolated,” wemean that the ECM is not in its conventional in vivo environment;rather, the ECM exists outside of the in vivo tissue with which isconventionally associated. Accordingly, isolated cardiac ECM is abioengineered cardiac ECM that is not located within myocardial hearttissue, and the term does not encompass naturally occurring cardiac ECM.

By “3-dimensional,” we mean that the ECM is not substantially flat(i.e., 2-dimensional), such that cells associated with the ECM may be incontact with ECM surfaces both above and below the cells. A3-dimensional ECM has a thickness of at least 20 μm.

By “cardiac,” we mean that the isolated ECM has a compositionsubstantially similar to, but not necessarily identical to, the in vivo3-dimensional extracellular matrix that is unique to cardiac tissue thatis undergoing healing after myocardial disease or injury. Substantialsimilarity is based on the type and abundances of the structuralproteins present in the ECM, as well as on the presence ofcharacteristic matricellular proteins, such as growth factors andcytokines. In the healing cardiac ECM, more than 60% of the structuralprotein molecules present are fibronectin molecules.

The cardiac ECM includes the structural proteins fibronectin, collagentype I, collagen type III, and elastin, and may include other structuralproteins as well. In some embodiments, the cardiac ECM includes thestructural protein collagen type V.

Preferably, fibronectin molecules make up from 60% to 90% of thestructural protein molecules present in the ECM. In some embodiments,fibronectin molecules make up from 70% to 90% of the structural proteinmolecules present in the ECM. In some embodiments, fibronectin moleculesmake up from 80% to 90% of the structural protein molecules present inthe ECM.

The bioscaffold that is made from the ECM has a thickness of 20-500 μm.In some embodiments, the bioscaffold has a thickness range of 30-200 μmor of 50-150 μm. In some embodiments, more than 80% of the structuralprotein molecules present are fibronectin molecules.

Preferably, the structural proteins of the cardiac ECM are notchemically cross-linked.

In addition to the structural proteins, the cardiac ECM may includematricellular proteins, such as growth factors and cytokines, as well asother substance. Non-limiting examples of other proteins that may befound in the cardiac ECM include latent transforming growth factor beta1 (LTGFB-1), latent transforming growth factor beta 2 (LTGFB-2),connective tissue growth factor (CTGF), secreted protein acidic and richin cysteine (SPARC), versican core protein (VCAN), galectin 1, galectin3, matrix gla protein (MGP), sulfated glycoprotein 1, protein-lysine6-oxidase, and biglycan. In some embodiments, the ECM may optionallyinclude one or more of transforming growth factor beta 1 (TGFB-1),transforming growth factor beta 3 (TGFB-3), epidermal growth factor-likeprotein 8, growth/differentiation factor 6, granulins, galectin 3binding protein, nidogen 1, nidogen 2, decorin, prolargin, vascularendothelial growth factor D (VEGF-D), Von Willebrand factor A1, VonWillebrand factor A5 A, matrix metalprotease 14, matrix metalprotease23, platelet factor 4, prothrombin, tumor necrosis factor ligandsuperfamily member 11, and glia derived nexin.

In certain preferred embodiments, the cardiac ECM is not attached to asolid surface, so that the bioscaffold can be readily used as anepicardial patch that can be applied to the surface of the heart wall.Optionally, the cardiac ECM is decellularized, and is thus essentiallydevoid of intact cardiac fibroblast cells. In some embodiments, thebioscaffold may be seeded using methods that are known in the art withone or more cells that are therapeutic for cardiac disease or injury.Examples of therapeutic cells types that could be used to seed thebioscaffold include without limitation skeletal myoblasts, embryonicstem cells (ES), induced pluripotent stem cells (iPS), multipotent adultgermline stem cells (maGCSs), bone marrow mesenchymal stem cells(BMSCs), very small embryonic-like stem cells (VSEL cells), endothelialprogenitor cells (EPCs), cardiopoietic cells (CPCs),cardiosphere-derived cells (CDCs), multipotent Is/1+ cardiovascularprogenitor cells (MICPs), epicardium-derived progenitor cells (EPDCs),adipose-derived stem cells, human mesenchymal stem cells (derived fromiPS or ES cells), human mesenchymal stromal cells (derived from iPS orES cells) skeletal myoblasts, or combinations thereof. All of these celltypes are well-known in the art.

Such embodiments also encompass methods for treating a subject having acardiac disease or injury, wherein the surface of the subject's heart iscontacted with a seeded epicardial patch as described above, and whereinthe severity of the cardiac disease or injury is decreased. Asdemonstrated in Examples 3 and 4 below, after being placed onto theheart surface, the patch adheres to the heart surface without the use ofglue, sutures, or other methods to facilitate attachment to the heartsurface. Thus, although the patch may be glued, sutured, or otherwiseattached to the surface of the heart, these steps are not necessary inperforming the method. The method can be generally used in the treatmentof cardiac disease or injury wherein cardiomyocytes are lost, includingwithout limitation in treating injury caused by an acute myocardialinfarct, by a bacterial infection, by a viral infection, by congenitaldefect, or by heart failure.

In a second aspect, the disclosure encompasses a method for preparing a3-dimensional cardiac extracellular matrix. The method includes thesteps of (a) isolating cardiac fibroblasts from cardiac tissue; (b)expanding the cardiac fibroblasts in culture for 1-7 passages; and (c)plating the expanded cardiac fibroblasts into a culture having a celldensity of 100,000 to 500,000 cells per cm². If the cell density is lessthan 100,000 cells per cm², the culture will fail to produce a3-dimensional ECM. Optionally, step (c) involves plating the expandedcardiac fibroblasts into a culture having a cell density of 100,000 to200,000 cells per cm².

The cardiac fibroblasts may be isolated using methods known in the art.Under these conditions, the cardiac fibroblasts secrete a 3-dimensionalcardiac extracellular matrix having a thickness of 20-500 μm that isattached to the surface on which the expanded cardiac fibroblasts areplated. Optionally, the cardiac ECM may have a thickness of 30-200 μm or50-150 μm.

In one embodiment, the cardiac fibroblasts are cultured in high glucoseDMEM+10% FBS and 1% penicillin/streptomycin at 37° in a 5% CO₂ 100%humidity atmosphere for 10-14 days. Optionally, after it is formed, thesecreted cardiac ECM may be contacted withethylenediaminetetraaceticacid (EDTA) to detach the cardiac ECM from thesurface on which it is plated. The detached cardiac ECM forms a freefloating bioscaffold that can seeded with therapeutic cells and used asan epicardial patch, as described previously.

The cardiac ECM formed in the method can optionally be decellularized toremove any remaining cardiac fibroblasts from the ECM. Anydecellularizing agent may be used to decellularize the cardiac ECM.Non-limiting examples of decellurizing agents known in the art includeenzymatic agents, such as trypsin, endonucleases, or exonucleases;chemical agents, such as alkaline or acid solutions, hypertonic orhypertonic solutions, ethylenediaminetetraacetic acid (EDTA), ethyleneglycol tetraacetic acid (EGTA), ammonium hydroxide, and peracetic acid;nonionic detergents, such as octylphenol ethylene oxide (Triton™-X 100);ionic detergents, such as sodium dodecyl sulfate (SDS) and polyethersulfonate (Triton™-X 200); and zwitterionic detergents, such as3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),sulfobetaine-10 and -16, and tri(n-butyl)phosphate.

In one embodiment, decellularization is performed by contacting thecardiac ECM with peracetic acid (PAA) and subsequently rinsing thecardiac extracellular matrix with water. Alternatively,decellularization is performed by contacting the secreted cardiacextracellular matrix with ammonium hydroxide (AH) and octylphenolethylene oxide (Triton™ X-100) and subsequently rinsing the cardiacextracellular matrix with water. Each method produces a differentcardiac ECM that can further be distinguished from the cardiac ECM whichis not decellularized, although all three cardiac ECMs contain a similarcomposition as defined by the structural proteins present.

Optionally, the method further includes the step of seeding the cardiacextracellular matrix with one or more cells that are therapeutic forcardiac disease or injury, using cell seeding methods that are wellknown in the art. Examples of such therapeutic cells include withoutlimitation skeletal myoblasts, embryonic stem cells (ES), inducedpluripotent stem cells (iPS), multipotent adult germline stem cells(maGCSs), bone marrow mesenchymal stem cells (BMSCs), very smallembryonic-like stem cells (VSEL cells), endothelial progenitor cells(EPCs), cardiopoietic cells (CPCs), cardiosphere-derived cells (CDCs),multipotent Is/1+ cardiovascular progenitor cells (MICPs),epicardium-derived progenitor cells (EPDCs), human mesenchymal stemcells (derived from iPS or ES cells), human mesenchymal stromal cells(derived from iPS or ES cells), or combinations thereof.

This aspect of the disclosure further encompasses a bioscaffold forfacilitating cell delivery to myocardial tissue (i.e. an epicardialpatch) that is made from the isolated 3-dimensional cardiac ECM producedby the method described above.

The following abbreviations and acronyms are used in this application:CF, cardiac fibroblast; CF-ECM, cardiac fibroblast-derived extracellularmatrix; ECM, extracellular matrix; EDTA,ethylenediamine-N,N,N′,N′-tetraacetic acid; DMAM, Dulbecco's modifiedeagle's medium; FBS, fetal bovine serum; FISH, fluorescence in situhybridization; hMSC, human mesenchymal stromal cells; MI, myocardialinfarction; PAA, Peracetic acid; PBST, phosphate buffer saline Tween®-20(Polyethylene glycol sorbitan monolaurate).

The following Examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and the following examples and fallwithin the scope of the appended claims.

EXAMPLES Example 1: Production and Characterization of Cardiac Derived3-Dimensional Extracellular Matrix

In this Example, we generated a 3D-ECM using cardiac fibroblasts that ismeasured by confocal microscopy to be between 30-150+μm thick.Additionally, we devised multiple ways to remove the fibroblasts(decellularization), which allow for the study of the effect of cellulardebris on stem cell attachment, proliferation and differentiation.Controlling (varying) the cell debris content is important becausecellular debris is highly prevalent in the early phases of cardiachealing, and has largely been overlooked as a factor impactingtherapeutic cell adhesion and differentiation. This new method ofgenerating cardiac specific extracellular matrix will facilitate invitro studies of stem cell interactions with a cardiac specific matrix.

An important first step in studying the CF 3D-ECM is carrying outdetailed analyses of its composition and structure. This was done usingbottom-up 2D-(strong cation exchange) mass spectrometry and confocalmicroscopy. Additionally, the effects of the matrix on cell morphology,proliferation and differentiation were studied using confocalmicroscopy, proliferation assay and quantitative PCR gene analysis.

Methods

Isolation of Cardiac Fibroblasts.

The technique for isolating cardiac fibroblasts is adapted frompreviously published reports (see Baharvand, H., et al., The effect ofextracellular matrix on embryonic stem cell-derived cardiomyocytes. JMol Cell Cardiol, 2005. 38(3): p. 495-503). Briefly, male Lewis rats(260-400 g) were sacrificed by CO₂ asphyxiation, hearts rapidly excised,atria removed and ventricles placed into ice cold PBS with 1%penicillin/streptomycin. Hearts were finely minced then placed into 10ml digestion media (DMEM, 73 U/ml collagenase 2, 2 μg/ml pancreatin(4×)) and incubated at 37° C. with agitation for 35 minutes. The digestmixture was centrifuged at 1000×g for 20 minutes at 4° C. The resultingcell pellet was suspended in 10 mls of fresh digestion media andincubated at 37° C. with agitation for 30 minutes. The resulting digestwas sieved through a 70 μm cell strainer and digest solution dilutedwith 10 ml of culture media (DMEM, 10% FBS, 1% penicillin/streptomycin).The cell suspension was then centrifuged at 1000×g for 20 minutes at 4°C. The cell pellet was suspended in 16 ml culture media and plated intotwo T75 culture flasks (8 ml per flask). The cells were allowed toattach under standard culture conditions (37° C., 5% CO₂, 100% humidity)for 2 hours, then non-adherent cells removed by washing with PBS andculture media replaced. Primary cardiac fibroblast cultures weretypically confluent in 4-7 days.

Generation of 3-Dimensional Cardiac Fibroblast Extracellular Matrix.

Cardiac fibroblasts (Passage 1-7) were plated at a density ofapproximately 1.1×10⁵ to 2.2×10⁵ per cm² in high glucose DMEM+10% FBSand 1% penicillin/streptomycin and cultured at 37° C., 5% CO₂ and 100%humidity for an average of 10±3 days. Removal of the cardiac fibroblastsfrom the matrix was done by several different methods, as explainedbelow.

Method 1: Matrix Coated Culture Surface (Ammonium Hydroxide/TritonX-100, “Clean Matrix”).

Cardiac fibroblasts were removed from the secreted extracellular matrixby incubation with 20 mM ammonium hydroxide+0.1% Triton™ X-100 (AHbuffer) for 24-48 hours at 4° C. with constant agitation. The resultingmatrix was then rinsed repeatedly with sterile water followed by PBS orculture media. For this method, the cardiac fibroblasts were plated ontocollagen type 1 coated dishes, which give the 3D-CF ECM a foundation toattach to so it will not lift off during decellularization.

Method 2: Matrix Coated Culture Surface (Peracetic Acid, “DirtyMatrix”).

Cardiac fibroblasts were removed from the secreted extracellular matrixby incubation with 0.15% peracetic acid (PAA buffer) for 24-48 hours at4° C. with constant agitation. The resulting matrix was then rinsedrepeatedly with sterile water followed by PBS or culture media. PAA doesnot remove the matrix from the surface of the plate, so collagen type 1was not required to anchor the matrix to the dish.

Method 3: Matrix “Patch.”

Cardiac fibroblasts and secreted extracellular matrix were removed fromthe culture dish as a single patch by incubation with 2 mM EDTA solutionat 37° C. The resulting patch was then decellularized with either Method1 or 2 described above.

Isolation of Bone Marrow Mesenchymal Stem Cells.

The technique for isolating bone marrow mesenchymal stem cells wasadapted from previously published reports (see Tropel, P., et al.,Isolation and characterisation of mesenchymal stem cells from adultmouse bone marrow. Exp Cell Res, 2004. 295(2): p. 395-406). Briefly,male Lewis rats (260-400 g) were sacrificed by CO₂ asphyxiation. Femursand tibias were bilaterally excised and soft tissue removed. The boneswere placed in ice cold PBS with 1% penicillin/streptomycin. In asterile culture hood, the ends of the bones were removed and an 18 gaugeneedle and syringe used to flush the shafts of the bones with culturemedia (DMEM, 10% FBS, 1% penicillin/streptomycin). The resulting bonemarrow was further dispersed by passage through a 21 gauge needle. Cellsuspension was then centrifuged at 1000×g for 10 minutes at 4° C. andplated into a 100 mm culture dish. The cells were allowed to attachunder standard culture conditions (37° C., 5% CO₂, 100% humidity) for 24hours, then non-adherent cells were removed by washing with PBS and theculture media was replaced.

Bottom Up Mass Spectrometry:

In-Solution Trypsin Digestion.

All solutions were prepared fresh just prior to use with HPLC gradewater. CF 3D-ECM patch was prepared using Method 3, as described above,and cut in half. The halves were decellularized by either Method 1 or 2.The resulting decellularized patch was suspended in 15 μl 8M Urea andthen 20 μl of 0.2% ProteaseMax™ added. The CF 3D-ECM was then dissolvedinto solution by vortexing and pipetting. 58.5 μl of 50 mM NH₄HCO₃ wasadded to a final volume of 93.5 The sample was then reduced by adding 1μl of 0.5 M DTT and incubating at 56° C. for 20 minutes. 2.7 μl of 0.55M iodoacetamide was added and incubated for 15 minutes at roomtemperature in the dark. 1 μl of 1% ProteaseMax™ and 2 μl of 1 μg/μlTrypsin Gold™ added and incubated overnight at 37° C. The following day,0.5 μl of trifluoroacetic acid was added to a final concentration of0.5% to stop the reaction. The sample was then centrifuged at 14,000×gfor 10 minutes at 4° C. and the cleared supernatant transferred to afresh 1.5 ml protease-free tube.

2D Liquid Chromatography Mass Spectrometry.

2 μl of sample was injected onto an Eksigent 2D nanoLC chromatographysystem and eluted into a Thermo Finnigan LTQ Mass Spectrometer. Thesample was retained on an Agilent Zorbax SB300-C8 trap and eluted byreverse phase gradient onto a 0.100 mm×100 mm emitter packed in-housewith 5 μm bead 300 angstrom pore MagicC18 material. Mobile phasesolution consisted of a water and 0.1% formic acid aqueous phase and a0.1% formic acid in 50% acetonitrile:ethanol organic phase. The gradientran from 1 to 60 minutes and from 5 to 35% organic with a 95% wash.Eluent was ionized by a positive 3000V nanoESI and analyzed by a DataDependent triple play template. The top 5 m/z were selected byintensity, charge state was analyzed by zoom scan, and MS/MS wereperformed with wideband activation, dynamic exclusion of 1 for 60seconds with a list of 300 m/z and a width of +/−1.5/0.5 m/z, collisionenergy of 35%, and noise level of 3000NL.

Sequest searches were performed via Bioworks 3.0 using a downloadedSwissprot database for Rat (October 2010) and its reversed sequences.Search parameters included trypsin digestion, 1 missed cleavage, aminoacid length of 6 to 100 with tolerance of 1.4 da, dynamic modificationsof methionine methylation (+14 da) and cysteine carboxyamidomethylation(+57 da). Results were filtered to less than 5% False Discovery Rate(FDR), defined by number of proteins identified with reversed sequencesdivided by the total number proteins identified minus reversed number,multiplied by 100.

Confocal Microscopy:

Paraffin Embedded CF 3D-ECM Patch Slides.

CF 3D-ECM patches were fixed in fresh 3.6% paraformaldehyde thenembedded in paraffin and sectioned in 5 μm sections and mounted onslides. Slides were deparaffinized by two incubations in xylene for 5minutes each followed by rehydration: 100% ethanol 2×5 minutes, 90%ethanol 2×5 minutes, 80% ethanol 1×5 minutes, 50% ethanol 1×5 minutes,water 2×5 minutes. A hydrophobic barrier was applied around the samplesand the slides incubated with 0.1% trypsin solution for 10 minutes at37° C. Slides were rinsed under running water and washed 2×5 minutes inPBS. A sodium citrate heat retrieval was performed by incubation in 10mM Sodium citrate, 0.05% Tween®-20 buffer pH 6 for 60 minutes in anOster® rice steamer (temperature approximately 95-100° C.). The stainingdish was removed from the rice steamer and the slides were cooled for 20minutes at room temperature then blocked with 1% bovine serum albumin inPBST for 1 hour at room temperature. Primary antibodies were added at adilution of 1:50 (all antibodies except when noted were purchased fromSanta Cruz Biomedical) and incubated at 37° C. for 1 hour. Slides werethen washed 3×5 minutes in PBST and incubated in secondary antibodies at1:1000 dilution in 1% bovine serum albumin in PBST (all secondaryantibodies purchased from Invitrogen) for 1 hour at room temperature inthe dark. Slides were then washed 2×5 minutes in PBST and counterstained with 1 μg/ml DAPI for 10 minutes followed by 1 wash in PBST anda final rinse in water. Cover slips were then mounted with aqueousmounting media and the edges sealed with quick dry, clear nail polish.Slides were imaged at the W.M Keck Laboratory for Biological Imagingwith a Nikon AIR scanning confocal microscope.

ECM Coated Coverslips.

Cardiac fibroblasts cultured on glass covers slips were decellularizedusing either Method 1 or 2 as discussed previously, then fixed with −20°C. methanol for 20 minutes. The cover slips were washed with 1% bovineserum albumin in PBST 3×5 minutes each, then blocked in 1% bovine serumalbumin in PBST for 1 hour at room temperature. Primary antibodies wereadded at a concentration of 1:50 and incubated overnight at 4° C. withgentle agitation. Cover slips were then washed 3×5 minutes in 1% bovineserum albumin in PBST. Secondary antibodies were added at 1:1000dilution and incubated for 1 hour at room temperature in the dark. Coverslips were then washed 2×5 minutes then counter stained with 1 μg/mlDAPI for 10 minutes at room temperature in the dark. Covers slips werethen washed with 1% bovine serum albumin in PBST for 5 minutes andrinsed in water, then mounted to slides with aqueous mounting media andsealed with fast dry nail polish. Slides were imaged at the W.M KeckLaboratory for Biological Imaging with a Nikon AIR scanning confocalmicroscope.

Proliferation Assay:

Bone marrow derived mesenchymal stem cells were isolated as describedpreviously. After expansion of the cells in culture, they were washed inPBS then trypsinized with 0.5% trypsin, 2.5 mM EDTA solution until freefloating. Cells were then centrifuged at 1000×g for 10 minutes at 4° C.The supernatant was removed and the cells suspended in DMEM+10% FBS and1% penicillin/streptomycin and counted on a hemocytometer using trypanblue. The cells were then seeded onto either tissue culture treatedplastic or decellularized matrix at a concentration of 1.0×10⁵ cells per40 mm dish. Cells were cultured for 5 days then trypsinized as describedabove and recounted on a hemocytometer. Statistical analysis was carriedout using a One-way ANOVA followed by a Tukey's post hoc test. p<0.05was considered statistically significant.

Quantitative Real-Time PCR Gene Analysis:

RNA Isolation and Purification.

RNA was isolated from cells using the Purelink RNA Isolation Kit®(Invitrogen) according to manufacturer's directions. Briefly, cells weretrypsinized as described above then pelleted by centrifugation at 1000×gfor 10 minutes at 4° C. Cells were washed in PBS, and pelleted as above,then moved to a fresh RNase-free 1.5 ml tube. Cells were suspended in300 μl of lysis buffer then aspirated through a 27½ gauge needle 15 to20 times. 300 μl of 70% ethanol was mixed with the sample by vortexingand pipetting before being transferred to a spin cartridge andcentrifuged at 12,000×g for 15 seconds at room temperature. Theflow-through was discarded and 700 μl of wash buffer 1 was placed ontothe spin cartridge and centrifuged at 12,000×g for 15 seconds at roomtemperature. 40 units of RNase free DNase 1 (Qiagen) were placed on thespin cartridge and incubated for 15 minutes at room temperature. 500 μlof wash buffer 1 was added to the spin cartridge and centrifuged at12,000×g for 15 seconds at room temperature. The spin cartridge waswashed twice as described above with two volumes of 500 μl wash buffer2. The spin cartridge was then dried by centrifugation at 12,000×g for 3minutes at room temperature and the RNA eluted from the spin cartridgeby incubating with 30 μl of RNase free water followed by centrifugationat 12,000×g for 1 minute at room temperature. RNA amount and 260/280absorbance ratio were read on a NanoDrop® ND-1000 spectrophotometer.

cDNA Synthesis.

Synthesis of cDNA was carried out using the RT² First Strand Kit(Qiagen) according to manufacturer's directions. Briefly, 475 ng of RNAwas mixed with 2 μl of Genomic DNA elimination buffer and volumeadjusted to 10 μl with RNase-free water. The RNA was then incubated for5 minutes at 42° C. then placed on ice. Reverse-transcription master mixwas made by mixing: 4 μl 5× buffer BC3, 1 μl control P2, 2 μl RE3reverse transcription mix, 3 μl RNase-free water (10 μl total) for eachsample. 10 μl of the reverse-transcription mix was added to each sampleand incubated at 42° C. for 15 minutes then the reaction was stopped byimmediately incubating the sample at 95° C. for 5 minutes. 91 μl ofRNase-free water was then added to each sample and the samples stored at−80° C. until analyzed. Before analysis on a Custom Rat RT² Profiler PCRArray, the quality of the sample was tested on a Quality Control (QC)plate testing for reverse transcription, positive PCR control, genomicDNA contamination, no reverse transcription and no template controls.

Custom Rat RT² Profiler PCR Array.

A commercially available Rat Mesenchymal Stem Cell Profiler PCR arraywas customized to include four markers of cardiac differentiation. Thekit was used according to manufacturer's directions. Briefly, 1350 μl 2×RT² SYBR Green Mastermix, 102 μl cDNA synthesis reaction and 1248 μlRNase-free water were gently mixed together by pipetting. The Profilerplate was loaded by adding 25 μl of the above reaction mix to each well.The plate was centrifuged at 1000×g for 3 minutes and cycled with thefollowing program: 1 cycle 10 minutes at 95° C., 40 cycles of 15 secondsat 95° C., 1 minute 60° C. (fluorescence data collection). Data wereanalyzed using software available from SABioscience and false discoveryrate was determined with the QVALUE package for R statistical software.

Results

Identification of Cardiac Fibroblasts.

In culture, fibroblasts often differentiate into myofibroblasts, ahighly proliferative regulator of extracellular matrix synthesis andmaintenance. These myofibroblasts are primarily responsible forgenerating granulation tissue after cardiac injury. First, we confirmedthe identity of the isolated cell population by staining for discoidindomain receptor 2 (DDR2), a cardiac fibroblasts specific marker. Second,to test if the cardiac fibroblasts had adopted a myofibroblastphenotype, we stained for α-actin, which is expressed in myofibroblastsbut not fibroblasts. The immunostaining assay demonstrated the robustexpression of DDR2 in the isolated cell population, indicating that thecell population was highly enriched for cardiac fibroblasts. 24 hoursafter plating, the immunostaining assay showed the co-expression ofα-actin and DDR2. This is consistent with a myofibroblast phenotype,suggesting the cells had adopted a highly proliferative state that isknown to synthesize large amount of extracellular matrix.

Generation of CF 3D-ECM.

Simple extracellular matrices preparations such as fibronectin, lamininand collagen are often referred to as 2-dimensional due to the thincoating (<1 micron) obtained on the culture surface. 3-dimensional cellculture has been shown to impart cellular morphologies and growthcharacteristics that are similar to those observed in vivo (see Justice,B. A., N. A. Badr, and R. A. Felder, 3D cell culture opens newdimensions in cell-based assays. Drug Discov Today, 2009. 14(1-2): p.102-7).

To investigate the use of 3-dimensional culture technologies incardioregenerative medicine, we developed two methods to denude cardiacfibroblasts from high density cultures in vitro. This resulted in theproduction of two distinct types of CF 3D-ECM. FIGS. 1A and B, whichshow a stained paraffin embedded matrix in a 5 μm section that isapproximately 45-50 μm thick, demonstrate representative examples ofMethod 1, decellularization by ammonium hydroxide/Triton X-100 (AH).Decellularization Method 2 employed peracetic acid (PAA) to denudecardiac fibroblasts from high density cultures in vitro.

Generation of CF 3D-ECM Patch.

The use of tissue constructs for therapeutic cell transfer incardioregenerative medicine is an area of intense research. The CF3D-ECM that we have generated could be used for this purpose. Wedeveloped a method to remove the 3-dimensional fibroblast/matrix layeras a continuous sheet using EDTA. The sheet can then be decellularizedusing either AH or PAA. In addition to CF 3D-ECM patches, thenon-decellularized cardiac fibroblast/matrix sheets can also be used asa viable patch option and are termed CF patches hereafter. FIG. 2 showsa representative CF patch. The patch produced in a 40 mm diameterculture dish contracts to approximately 20 mm in diameter after releasefrom the plate.

Proteomics Based Analysis of CF 3D-ECM Structural and MatricellularProteins:

Structural Protein Composition of CF 3D-ECM.

Fibroblasts synthesize a matrix unique to the tissue in which theyreside. For this reason, we used 2D-mass spectrometry to evaluate thestructural and matricellular proteins composing the matrix. Structuralproteins were considered to be the extracellular proteins fibronectin,collagens, and elastin. We determined the breakdown of known structuraland matricellular (non-structural proteins associated with the matrix)proteins as well as proteins that are neither structural normatricellular (other) proteins found in AH and PAA decellularizedmatrix. Specifically, in the AH/Triton decellularized matrix, 31% of theproteins by total spectral counts were structural, 2% werematricellular, and 67% were neither (other). In the PAA decellularizedmatrix, 9% of the proteins by total spectral counts were structural, 1%were matricellular, and 90% were neither (other).

We further determined the distribution of structural proteins in the AHand PAA matrices. In the AH/Triton decellularized matrix, 89% of thestructural protein by total spectral counts was fibronectin, 7.8% wascollagen type 1, 2.0% was collagen type 3, and 0.9% was elastin. In thePAA decellularized matrix, 88.7% of the structural protein by totalspectral counts was fibronectin, 8.3% was collagen type 1, 2.2% wascollagen type 3, and 0.52% was elastin. While the two decellularizationtechniques created uniquely different matrices in terms of relativeamounts of structural, matricellular, and non-structural/matricellularproteins, they did not significantly alter the relative amounts ofstructural proteins present in the matrix.

Analysis of Matricellular Proteins Contained in CF 3D-ECM.

The extracellular matrix is known to be a repository for bioactivemolecules such as growth factor and cytokines. Such extracellular matrixassociated bioactive molecules are termed matricellular proteins and areimportant for cell adhesion, proliferation, and differentiation. Toidentify the unique, low abundance bioactive molecules, Bottom-Up2D-mass spectrometry was used. Table 1 details the growth factors andcytokines that were detected in the matrix. Thirty proteins wereidentified between the two decellularization conditions by bottom-up 2Dmass spectrometry. Twenty-one matricellular proteins were identified inthe PAA samples and twenty-three in AH matrix sample.

To confirm the phenotype of the isolated BMSC, we stained for surfacemarkers CD29, CD44, and c-kit (FIGS. 3A, B, and C respectively). Asexpected, the BMSC expressed all three markers.

BMSC Morphology in CF 3D-ECM.

Cell morphology in 3-dimensional culture has been shown to be more invivo like than 2-dimensional culture (see Justice, B. A., N. A. Badr,and R. A. Felder, 3D cell culture opens new dimensions in cell-basedassays. Drug Discov Today, 2009. 14(1-2): p. 102-7). To determine howculture of BMSC on CF 3D-ECM affects cell morphology, BMSC were platedonto either glass coverslips or coverslips coated with CF 3D-ECM. 48hour after plating, cell morphology was examined with confocalmicroscopy. FIG. 4A demonstrates a typical in vitro fibroblastic BMSCmorphology. FIG. 4B demonstrates that cells cultured on CF 3D-ECM takeon an elongated morphology, aligning with the CF 3D-ECM.

Proliferation of BMSC on CF 3D-ECM.

The surface on which a cell is cultured impacts the ability and/or rateat which the cell proliferates. With our detection of extensivematricellular proteins in the CF 3D-ECM it would seem reasonable thatproliferation of BMSC would be affected. To determine the effect of CF3D-ECM on proliferation, we performed a proliferation assay and foundthat culturing cells on CF 3D-ECM approximately doubled theproliferation rate of BMSC compared to those cultured on tissue culturetreated plastic. Of the two matrix conditions, the “clean” AH matrixincreased cell proliferation significantly more than PAA, 263% and 193%respectively compared to plastic (FIG. 5).

It has been established that the surface/matrix a stem cell is culturedon impacts the differentiation state of the cell (see Santiago, J. A.,R. Pogemiller, and B. M. Ogle, Heterogeneous differentiation of humanmesenchymal stem cells in response to extended culture in extracellularmatrices. Tissue Eng Part A, 2009. 15(12): p. 3911-22). To determine howthe cardiac specific AH and PAA matrices effected gene expression ofBMSC, including markers of stemness, mesenchymal specificity, anddifferentiation, we carried out an experiment according to FIG. 6. Wepassaged primary (P0) BMSC onto tissue culture plastic (P1), AH matrix,or PAA matrix (FIG. 6). Specifically, BM-derived MSCs were cultured for5 days on plastic. The ‘Passage 0’ group was then removed and mRNA wascollected. The ‘Passage 1’ group was passaged onto plastic, CF-derivedmatrix that was decellularized with ammonium hydroxide, or CF-derivedmatrix that was decellularized with PAA, and cultured for another 5 daysbefore removal and collection of mRNA. As expected, passage alone hadsignificant effects on BMSC gene expression. The expression of nineteengenes was altered by more than two-fold, with the expression of sixgenes increasing and thirteen genes decreasing (data not shown).

Interestingly, passage onto AH or PAA-treated matrix had significantlydifferent effects in comparison to the cells cultured on tissue cultureplastic (P1). Specifically, the AH matrix significantly altered theexpression of twenty-three genes by greater than two-fold, with theexpression of nine genes increasing and the expression of fourteen genesdecreasing (data not shown). The PAA matrix significantly altered theexpression of seven genes by greater than two-fold, with the expressionof three genes increasing and the expression of four genes decreasing(data not shown). Twenty-four genes were significantly differentiallyexpressed in BMSC cultured on PAA relative to AH matrix (data notshown). This finding is interesting, and may shed light on the effectsof such factors as cell debris and matricellular proteins in cellattachment, proliferation, and gene expression.

Discussion

In this Example, we set out to 1) use novel culture techniques to inducecardiac fibroblasts to produce a 3D matrix that models phases of cardiachealing; 2) determine how different methods of fibroblast removal affectthe protein composition of the 3D-ECM; and 3) determine how BMSC areaffected by culture on 3D ECM of different protein compositions.

Using Novel Culture Techniques to Induce Cardiac Fibroblasts to Producea 3-Dimensional Matrix that Models Phases of Cardiac Healing.

In vivo, cells residing within any organ exist in a 3-dimensionalenvironment. Work by Cukierman et al. and others have demonstrated theprofound effects that culturing cells in a 3-dimensional matrix can haveon important cellular characteristics such as adhesion, proliferation,and migration (Cukierman, E., et al., Taking cell-matrix adhesions tothe third dimension. Science, 2001. 294(5547): p. 1708-12).

Early attempts by VanWinkle to create a cardiac specific extracellularmatrix resulted in deposition of extracellular matrix proteins onto theculture surface (VanWinkle, W. B., M. B. Snuggs, and L. M. Buja,Cardiogel: a biosynthetic extracellular matrix for cardiomyocyteculture. In Vitro Cell Dev Biol Anim, 1996. 32(8): p. 478-85). Thematrix deposited using the VanWinkle technique was extremely thin, onthe order of <0.1 μm in total thickness. A matrix of this thicknessshould not be considered 3-dimensional because it does not create anenvironment where cells can interact on all sides. In contrast, we wereable to create cardiac specific matrix that is consistently >50 μm inthickness, with some preparations growing as large as 150+μm.

TABLE 1 Matricellular proteins found in CF 3D-ECM* Spectral Hits PAA AHProliferation/Differentiation Transforming growth factor beta 1 0 1Transforming growth factor beta 3 1 4 Latent transforming growth factorbeta 1 2 2 Latent transforming growth factor beta 2 2 10 Connectivetissue growth factor 4 18 Epidermal growth factor-like protein 8 0 1Growth/differentiatoin factor 6 0 1 Granulins 5 0 SPARC 14 13 VersicanCore Protein 13 63 Adhesion Galectin 1 11 2 Galectin 3 21 23 Galectin 3binding protein 3 0 Nidogen 1 19 0 Nidogen 2 0 1 Decorin 0 1 Prolargin 19 Angiogenesis Vascular endothelial growth factor D 1 0 VonWillebrandfactor A1 0 2 VonWillebrand factor A5 A 9 0 Proteases MatrixMetaloprotease 14 1 0 Matrix Metaloprotease 23 0 1 Other MatricellularProteins Matrix Gla Protein 6 13 Sulfated glycoprotein 1 46 2 Plateletfactor 4 0 1 Protein-lysine 6-oxidase 2 4 Prothrombin 1 2 Tumur necrosisfactor ligand superfamily member 11 0 1 Biglycan 15 15 Glia derivednexin 1 0 *Relative abundance of the proteins may be indicated by thenumber of spectral hits. Reliable quantification of the matricellularproteins based in this data is difficult, due to their low abundance.

We discovered that when cardiac fibroblasts were isolated and culturedthey responded with an “injury” pattern of extracellular matrixproduction, synthesizing a matrix high in fibronectin, with lessercomponents including collagen types 1 and 3. This phenomenon allowed usto model some of the phases of cardiac healing. The composition of thestructural proteins in CF 3D-ECM (Table 1) is similar to that of asecond order matrix formed during cardiac healing process. Studies inthe infarcted canine myocardium found that the second order matrix isprimarily composed of fibronectin (64%) with small amounts of collagentypes 1 and 3 (Dobaczewski, M., et al., Extracellular matrix remodelingin canine and mouse myocardial infarcts. Cell Tissue Res, 2006. 324(3):p. 475-88). In that study, fibronectin content peaked between 7-14 dayspost myocardial infarction.

While our data from isolated rat cardiac fibroblasts yielded a matrixwith somewhat more fibronectin (89%) than in the canine, the overallcomposition of the CF 3D-ECM was more similar to second order matrix,formed during phase three of cardiac healing, than either normal cardiacextracellular matrix or a mature scar. Because of these structuralsimilarities, we believe that we have created a matrix that models the7-14 day period post myocardial infarction. This time frame may be idealfor therapeutic cell therapy, because of reduced inflammatory response.Furthermore, due to its high fibronectin content, such a matrix may bemore conducive to cell adhesion than a mature scar with fully crosslinked collagen as the primary component. Thus, our CF 3D-ECM holdspromise as a reagent for the study of stem cell-ECM interaction withinthe healing myocardial extracellular matrix.

Determining how Different Methods of Fibroblast Removal Affect theProtein Composition of the 3D-ECM.

Cell debris is a natural component of the healing myocardium, especiallyin phases 1 (cardiomyocyte cell death) and 2 (acute inflammation). Todate, the effects of cell debris on therapeutic cell adhesion,proliferation, and differentiation has gone relatively unstudied. Wediscovered that while the method of decellularization (AH or PAA) didnot affect the composition of structural proteins (fibronectins,collagens, and elastin) in the matrix, it did effect the overall proteincomposition. PAA contained considerably more non-structural proteinscompared to AH matrix, demonstrating that PAA creates a matrix withsignificantly more cell debris than decellularization with AH.

It is important to note that other proteins that are not structural ormatricellular compose a large proportion of CF 3D-ECM. This may be tosome degree an artifact of the method used to characterize and quantifyrelative proportions of extracellular matrix (structural) proteins. Forexample, to perform bottom up 2D-mass spectrometry one must firstperform an in-solution trypsin digestion to produce peptides which canbe sequenced by the instrument. This proved to be difficult onextracellular matrix proteins, due to their low solubility in solution,structural complexity, and resistance to degradation by trypsin. It ispossible that non-structural matrix proteins were preferentiallydigested. Additionally, bottom up 2D-mass spectrometry is highlysensitive; we may be making a proverbial “mountain out of a mole hill”,with the ability to detect even the most minor of proteins present inthe matrix. Regardless of whether the high proportion ofnon-structural/matricellular proteins in CF 3D-ECM is a methodologicalartifact, the PAA decellularized CF 3D-ECM contained significantly morenon-structural/matricellular proteins than the AH decellularized CF3D-ECM, thus we consider the PAA matrix to be relatively “dirty” and theAH matrix to be relatively “clean”.

We demonstrated that PAA and AH create two distinctively differentmatrices in regards to the amount of cell debris contained in/on the CF3D-ECM, while maintaining the same extracellular matrix proteinstructural composition. The two distinctly different matrices resemblemore specific phases of cardiac healing. PAA, with its greaterproportion of cell debris, is more similar to phases 1 (cardiomyocytecell death) and 2 (acute inflammation) of cardiac healing, while AH,with its relatively cleaner matrix, may more closely resemble phase 3(granulation tissue formation).

Determining how BMSC are Affected by Culture on 3D ECM of DifferentProtein Compositions.

It has been well established that extracellular matrix has profoundeffects on cell morphology, proliferation, and differentiation(Cukierman, E., et al., Taking cell-matrix adhesions to the thirddimension. Science, 2001. 294(5547): p. 1708-12). In our CF 3D-ECM, alikely cause of these matrix effects on cells is the presence of thirtyspecific matricellular proteins we identified by bottom up 2D massspectrometry (Table 1). Ten of the thirty matricellular proteinsidentified are involved in cellular proliferation and differentiation.Several members of the transforming growth factor beta (TGF-β) superfamily were present in either active or latent form. TGF-β is known tobe secreted by myofibroblasts and promotes cell proliferation.Consistent with this, we found that BMSC grown on CF 3D-ECM had markedlygreater cellular proliferation (AH 263%, PAA 193%) compared to cellscultured on tissue culture treated plastic. Interestingly, proliferationwas significantly greater on “clean” AH matrix compared to the “dirty”PAA matrix (p<0.05). This could be the result of cell debris limitingcell-extracellular matrix interactions. The higher level of cell debrisin the PAA model may limit the cells access to the matricellularproteins, such as TGF-β, that promote proliferation.

Stem cells differentiate in response to passage in culture and toexposure to extracellular matrix. RT qPCR analysis of BMSC cultured ontissue culture plastic or CF 3D-ECM confirmed that both passage and CF3D-ECM affected the cells' differentiation state as indicated by changesin gene expression. We did not observe a clear up or down regulation ofa single differentiation pathway, likely due in large part to using anunselected heterogeneous BMSC cell population. Several firm conclusionscan be made. First, AH and PAA decellularized matrices are verydifferent. This is evident by the fact that of the 28 genes that had agreater than 2-fold difference in expression from P1 (passaged ontoplastic), not a single gene was changed equally by both AH and PAA.Additionally, AH matrix may prevent some passage-induceddifferentiation. AH matrix completely reversed the passage effect of 5genes (Adipoq, GDF5, BMP6, GDF7 and Tbx5). Finally, AH and PAA matricesdo not promote cardiomyogenic differentiation. Only one cardiac gene wasaltered by any condition; troponin T expression was decreased 2-fold byAH matrix.

This work represents initial studies of the effect of CF 3D-EMC on apotentially therapeutic cell population (BMSC). We determined how MSCare affected by culture on 3D ECM of different protein compositions byevaluating proliferation and gene expression associated with mesenchymalstem cells. While CF 3D-ECM clearly accelerated the proliferation rate,analysis of gene expression associated with mesenchymal stem cells wasmore complex. It is clear that CF 3D-ECM did not cause an adoption of acardiac phenotype. While the meaning of the gene changes is difficult tointerpret, it is clear that even a relatively short culture time on CF3D-ECM can induce BMSC to change phenotypes.

Summary.

We successfully generated a novel cardiac fibroblast derived3-dimensional matrix (CF 3D-ECM) that has characteristics similar to thematrix synthesized during cardiac healing. Using two specific methods todecellularize the matrices, we were able to generate matrix with varyingamounts of cell debris. The two distinctly different matrices inducedlarge changes in proliferation rate and BMSC gene expression. This novelCF 3D-ECM holds promise as both a reagent for use in studyingcell-matrix interactions (see Example 2) and as a bioscaffold “patch”for therapeutic cell transfer to the heart (see Examples 3 and 4).

Example 2: Dynamic Adhesion of Mesenchymal Stem Cells to CardiacFibroblast 3-Dimensional Extracellular Matrix

Therapeutic cell adhesion and engraftment are essential to the successof cell-based therapy. Current techniques involving circulatory infusion(systemic or intracoronary) have been used in large scale clinicaltrials with mixed results, with extremely low reported retention ratesfor both systemic and intracoronary infusion. Intramyocardial injection(either epicardial or endocardial) tends to have greater cell retentionrates than circulatory delivery modes and has the ability to delivercells with limited extravasation potential, such as skeletal myoblasts.To date, studies quantifying cell retention in vivo have been difficultand expensive to perform.

To address this issue, we utilized an oscillatory dynamic adhesion assaydeveloped by others to test the adhesion of BMSC to our CF 3D-ECMgenerated with AH or PAA and to cardiac fibroblasts(non-decellularized). We also developed a double ended antibody to CD44and fibronectin in an attempt to increase adhesion of BMSC to CF 3D-ECM.We demonstrated that adhesion to CF 3D-ECM was impacted by the method ofdecellularization. Specifically, it appears that the increased presenceof cell debris in the “dirty” PAA CF 3D-ECM significantly reduced BMSCadhesion to that matrix. Additionally, the presence of cardiacfibroblasts significantly reduced the ability of the BMSC to adhere tothe underlying matrix. Finally, we failed to alter cell adhesion using atargeted antibody tethering approach.

We combined the microfluidics oscillatory adhesion assay developed inanother lab with our CF 3D-ECM by culturing cardiac fibroblasts withinthe microchannels of a microfluidic device. Advantages of using thisassay system include: 1) adhesion to a cardiac specific extracellularmatrix can be measured quickly and is reproducible; 2) the oscillatorymotion of the dynamic adhesion assay is similar to the pulsatile natureof cardiac tissue; 3) a limited number of cells are needed to carry outquantitative studies (in traditional continuous flow adhesion assays,large numbers of cell are needed to maintain a continuous flow); 4) TheCF 3D-ECM can be manipulated to mimic the temporal aspect of cardiachealing using multiple decellularization techniques; and 5) treatmentstargeted at increasing therapeutic cell adhesion can be carried out,such as the antibody tethering experiments presented in this Example. Wetested three preparations of CF 3D-ECM in an attempt to mimic differentphases of cardiac healing.

Phase 1 and 2 (Cardiomyocyte Death and Acute Inflammation).

One feature of early cardiac healing is the presence of large amount ofcellular debris infesting the injured area. The presence of zones ofdense cell debris are termed contraction bands. To date, cell debris haslargely gone unstudied as a factor affecting therapeutic cell adhesion.Given the importance of cell-extracellular matrix interaction duringadhesion, any inhibition of receptor binding interactions could havelarge effects on the ability of a potentially therapeutic cell to bindto the matrix.

Phase 3 and 4 (Granulation Tissue Formation and Scar Remodeling/Repair).

Granulation tissue formation is initiated once macrophages remove amajority of the dead cells and debris from the injured area. Matrixdeposition during healing is an orderly process. A first order matrix iscomposed of primarily fibrin from the blood. This is replaced by asecond order matrix secreted by myofibroblasts infiltrating the damagedtissue. This second order matrix is composed primarily of fibronectinwith smaller amounts of collagen types I and III. The second ordermatrix serves as a scaffold for the deposit of collagen type III, whichis more rapidly produced, followed by collagen type I. Eventually, themature scar is composed of primarily collagen type I with smaller amountof collagen type III and virtually no fibronectin (Dobaczewski, M., etal., Extracellular matrix remodeling in canine and mouse myocardialinfarcts. Cell Tissue Res, 2006. 324(3): p. 475-88). A distinctivefeature of the cardiac scar is that the collagen is completelycross-linked, which adds strength to the scar.

A scar does not support large-scale infiltration or engraftment oftherapeutic cells, primarily because of the collagen cross-linking andpoor circulation. For this reason the scar has largely been abandonedduring targeted regenerative medicine treatments. Instead, efforts havefocused on the border zone, which contains hibernating cardiomyocytes,better circulation, and a more typical extracellular matrix.

Understanding the spatial and temporal aspects of cardiac healing allowsus to create an in vitro environment that more closely resembles theinjured myocardium during the various phases of cardiac healing. This invitro modeling is important to efficiently study therapeutic celladhesion and targeted treatments to increase engraftment in vivo.

To investigate the adhesion of therapeutic cells to extracellularmatrix, we have created three matrix conditions that resemble differentphases of cardiac healing. 1) Phase 1 and 2: Peracetic acid was used asa decellularizing agent. This method leaves large quantities of celldebris on the extracellular matrix. 2) Phase 3: Ammoniahydroxide-Triton™ X-100 is used as a decellularizing agent. This methodbetter removes cellular debris, leaving the extracellular matrixproteins exposed. 3) Late phase 3, beginning of phase 4: High densitycardiac fibroblast culture was used to resemble the peak ofmyofibroblasts infiltration. This condition was not decellularized in anattempt to more closely model the scar before a large proportion of themyofibroblasts undergo apoptosis.

Targeted treatments to increase therapeutic cell adhesion in the threeconditions were carried out using a novel strategy utilizing a doubleended antibody that binds to both CD44 and fibronectin. CD44 is highlyexpressed on BMSC and fibronectin is the major structural component tothe CF 3D-ECM, thus, adhesion should be increased unless the antibody isnot able to interact with fibronectin binding sites.

Methods

Generation of CF 3D-ECM.

Cardiac fibroblasts were isolated as described in Example 1. Cardiacfibroblasts were seeded at a confluent density into the microfluidicsdevices (FIG. 7), which were provided by the David Beebe lab at theWisconsin Institute for Medical Research at the University ofWisconsin-Madison. Fibroblasts were cultured in DMEM+10% FBS and 1%penicillin/streptomycin under standard culture conditions (37° C., 5%CO₂ and 100% humidity) for 2 hours, then the open ports were scrapedfree of cells to isolate the CF 3D-ECM in the device channel. Cardiacfibroblasts were further cultured for 10±3 days before decellularizationusing Method 1 or 2 as described in Example 1. Followingdecellularization, the CF-3D-ECM was washed repeatedly with PBS followedby 3 washes with serum free DMEM. All experiments were carried out usingserum-free DMEM.

Bone Marrow Mesenchymal Stem Cell Isolation.

Bone marrow mesenchymal stem cells (BMSC) were isolated 5-7 days beforethe experiment using the method in Example 1. The day of the experiment,BMSC were trypsinized, and split into tethered and non-tetheredfractions. Non-tethered BMSC were maintained on ice in serum-free DMEMuntil the experiment.

Antibody Tethering:

CD44 Antibody-Streptavidin Conjugation.

Anti-CD44 antibody from Santa Cruz (10 μg (50 μl)) was linked tostreptavidin with the Lightning-Link™ Streptavidin Conjugation Kit(Innova Biosciences) according to manufacturer's directions. Briefly, 5μl of LL-Modifier reagent was added to 50 μl of anti-CD44 antibody andmixed gently. Anti-CD44 antibody with LL-Modifier was pipetted directlyinto 10 μg of lyophilized Lightning-Link material and mixed bypipetting. The mixture was incubated overnight at room temperature. 5 μlof LL-quencher reagent was added to 50 μl of streptavidin linkedantibody and incubated for 30 minutes at room temperature. Streptavidinconjugated antibody was stored at 4° C. until use.

Bone Marrow Mesenchymal Stem Cell-Antibody Tethering.

Bone marrow mesenchymal stem cells were trypsinized as described aboveand suspended in 90 μl 2% FBS in PBS. 10 μl of streptavidin-linked CD44antibody was added and incubated at 4° C. for 30 minutes. Cells werewashed with 1 ml 2% FBS in PBS, centrifuged at 1000×g for 5 minutes at4° C. The antibody labeled cells were then suspended in 90 μl 2% FBS inPBS and 1.5 μg (10 μl) of biotin conjugated anti-rabbit antibody addedand incubated at 4° C. for 30 minutes. The cells were washed with 1 ml2% FBS in PBS, centrifuged at 1000×g for 5 minutes at 4° C. The cellswere suspended in 90 μl 2% FBS in PBS and 10 μl rabbit anti-fibronectinantibody added and incubated at 4° C. for 30 minutes. The cells werewashed with 1 ml 2% FBS in PBS, centrifuged at 1000×g for 5 minutes at4° C. Bone marrow mesenchymal stem cells tethered to anti-fibronectinantibody were then suspended in 1 ml serum free DMEM and stored on iceuntil use in the dynamic adhesion assay.

Oscillatory Dynamic Adhesion Assay.

The microfluidics device developed in the Beebe lab consists of two openports connected by a microchannel (FIGS. 7B and 7C). This configurationcreates passive pumping through the microchannel, allowing for cultureof cardiac fibroblasts and the generation of CF 3D-ECM directly in themicrochannel (FIG. 7A). To conduct the assay, a diaphragm is used toseal one set of ports, then a cantilever piezoelectric actuator isplaced directly over the center of the port in contact with thediaphragm. Voltage applied to the piezoelectric cantilever deflects thediaphragm, displacing the fluid under the diaphragm and resulting inshear-stress in the channel.

A pilot study was performed measuring BMSC adhesion to AH matrix, tissueculture treated plastic (TCP), and TCP+fibronectin under dynamicconditions. BMSC adhered more rapidly to AH matrix than to TCP orTCP+fibronectin (data not shown). Given the large difference in adhesionbetween TCP, TCP+fibronectin and the AH matrix, we determined that theassay would be appropriate for testing BMSC adhesion to CF 3D-ECM.Dynamic adhesion was measured for AH, PAA, and CF. BMSC adhered to AH ata significantly higher shear stress than PAA (p<0.003) or CF (p<0.0005).(n=3 animals)

Results

Dynamic BMSC Adhesion to AH-, PAA-CF 3D-ECM, and Cardiac Fibroblasts.

To test cell adhesion, we measured the 150 (the shear-stress at which50% of cells are adhered) of BMSC to either AH matrix, PAA matrix, orcardiac fibroblasts (CF). We found that BMSC adhered at a significantlyhigher shear-stress to the AH matrix than PAA matrix (p<0.003) or CFs(p<0.0005). BMSC isolated from three animals were tested for eachcondition, consisting of at least three microchannels per animal percondition (data not shown).

Effects of Tethering Anti-Fibronectin Antibody to BMSC.

The cardiac specific oscillatory dynamic adhesion assay allows for thescreening of methods targeted at increasing therapeutic cell adhesion inan in vitro system. We tested the potential for the double endedCD44-fibronectin antibody to affect adhesion to either AH, PAA-matrix,or CF. We found that adhesion of BMSC was not affected by the presenceof the double ended CD44-fibronectin antibody (data not shown). BMSCisolated from three animals were tested for each condition, consistingof at least three microchannels per animal per condition.

Discussion

This Example was designed to 1) determine if BMSC adhesion was affectedby the three matrix preparations; and 2) use the oscillatory dynamicadhesion assay in conjugation with our 3-dimensional ECM to test atargeted approach to increasing cell adhesion.

Determining if BMSC Adhesion was Affected by the Three MatrixPreparations.

Cardiac healing is a progressive event composed of specific phases. Eachphase is accompanied by changes in extracellular matrix structure andcomposition. We created two different CF 3D-ECMs using PAA or AH and athird non-decellularized condition (cardiac fibroblasts). As describedin Example 1, the composition of structural proteins in both AH and PAAmatrix is similar to that of the second order matrix formed duringgranulation tissue formation, but the amount of cellular debris issignificantly different between the AH and PAA matrix preparations.Using these two different matrix preparations and cardiac fibroblasts,we were able to show that BMSC adhere under a greater shear stress to a“clean” (AH) matrix compared to the “dirty” (PAA) matrix or CFs.

These findings suggest a window of opportunity for therapeutic celltransfer that may be most amenable to adhesion. We speculate that windowmay be between 7-14 days post infarction. During this time theinfiltrating macrophages have removed most of the dead cells and debris,while myofibroblasts have produced the fibronectin rich second orderscar. Injecting therapeutic cells before this time may reduce adhesiondue to increased cell debris, while waiting to inject therapeutic cellsafter the 7-14 day window may result in reduced adhesion because ofpeaking numbers of myofibroblasts and the formation of a mature scar.

We recognize that the conditions tested do not fully recapitulate ahealing myocardium. There are several components not accounted for inthis model, such as the presence of immune cells. Additionally, to ourknowledge, the shear-stress felt by a cell injected into the myocardiumis not known. For that reason, we used a technique called a “sweep”.This technique slowly reduces the shear-stress of the assay resulting inthe ability to detect adhesion across several orders of magnitude ofshear-stress.

Using the Oscillatory Dynamic Adhesion Assay to Test a Targeted Approachto Increasing Cell Adhesion.

We attempted to affect BMSC adhesion to the CF 3D-ECM matrix bytethering BMSC with a double ended CD44-Fibronectin antibody. We wereunable to detect a difference in the adhesion rates of the antibodytethered cells compared to the control cells under any condition. Thereare several possible explanations for this finding. For example, thesize of the cell in comparison to the antibodies may have been toogreat, creating a condition where the antibody was not long enough toreach the fibronectin of the matrix or the antibody interactions notstrong enough to increase adhesion. Additionally, the availability offibronectin for the antibody to interact with could be impacted byspatial organization of the extracellular matrix components of CF 3D-ECMor the amount of cell debris. Finally, we cannot rule out thepossibility that the CD44-fibronectin antibody failed to form correctly,as we had no means to assess this. Less likely is the possibility of theantibodies being internalized over the several hours it takes to performthe assay, because we observed no difference in adhesion rates from thebeginning to the end of the assay.

Summary.

We were successful in growing CF 3D-ECM in microfluidic channels whereBMSC adhesion could be studied under dynamic conditions. We demonstratedthat adhesion to CF 3D-ECM was impacted by the method ofdecellularization. We speculate that the increased presence of celldebris in the “dirty” PAA CF 3D-ECM significantly reduced BMSC adhesionto the matrix. Additionally, the presence of cardiac fibroblasts alsosignificantly reduced the ability of the BMSC to adhere to theunderlying matrix. Finally, we failed to demonstrate an increase in celladhesion with a targeted antibody tethering approach.

Example 3: Cardiac Fibroblast Patches: Successful Adhesion of EpicardialPatch to Epicardium

Epicardial patches have emerged as a promising mode of therapeutic celldelivery for cardiac regeneration. One issue with using synthetic ordecellularized tissues as patch materials is the inability of the patchto physically adhere to the surface epicardium, often requiring the useof glue or sutures to hold the patch to the heart. If the patch does notmaintain firm contact with the surface of the heart, the ability of thecells to transfer is decreased significantly. We demonstrate in thisExample that CF 3D-ECM adheres well to the surface of a heart, creatinga unique cardiac based platform for therapeutic cell transfer. This workrepresents collaboration with the laboratory of Professor Brenda Ogle ofthe Stem cell and Regenerative Medicine Center at the University ofWisconsin-Madison. Nicholas Kouris of Professor Ogle's laboratoryprovided human mesenchymal stem cells and carried out the CD73 stainingand cell counts.

The CF 3D-ECM we have created is cardiac specific, and its proteincomposition closely mimics a second order matrix formed duringgranulation tissue formation. The patch is predominantly composed offibronectin, which is considered an adhesive component of theextracellular matrix. Because of this high fibronectin content, wehypothesized that CF 3D-ECM would adhere well to the surface of a heart,creating a unique cardiac platform for therapeutic cell transfer.

To test this, a mouse myocardial infarction model was used. Epicardialpatches composed of either PAA decellularized CF 3D-ECM or anon-decellularized patch were seeded with embryonic stem cell derivedhuman mesenchymal stem cells (hMSC). Patches were placed on the surfaceof the infarcted myocardium 24 hours after infarction and subjectivelyevaluated for initial adhesiveness by a blinded impartial third partywho has experience placing epicardial patches. Mice were sacrificed 48hours after patch placement and hearts examined for sign of cardiacpatch adhesion. Histology (H&E) was used to detect patches and confocalmicroscopy used to detect CD73 express in the patch and myocardium.

Methods

Generation of CF 3D-ECM Patches.

The method for CF-3D-ECM patch production is detailed in Example 1. Thenotable difference is the use of both a PAA decellularized patch (“CF3D-ECM patch”) and a patch that is only lifted off the plate with 2 mMEDTA and not decellularized (cardiac fibroblasts left in patch; “CFpatch”).

Seeding of Human Mesenchymal Stem Cells (hMSC) onto Patches.

For this experiment two types of patches were used, PAA decellularizedand cardiac fibroblast (CF) patches. Decellularized patches wereincubated with PAA overnight at 4° C., and then rinsed repeatedly inPBS+1% penicillin/streptomycin. CF patches were removed from the platejust prior to seeding with hMSC. Both PAA CF 3D-ECM and CF patches wererinsed in serum-free DMEM and trimmed to approximately 1 cm squares andseeded with 5×10⁵ hMSC in 5 μl serum-free DMEM for 2 hours prior toplacement on the heart.

Myocardial Infarction Model.

Following induction of isoflurane anesthesia (3%), the mouse wasintubated with an 18 gauge catheter and placed on a mouse ventilator at120-130 breaths per minute with a stroke volume of 150 μl and maintainedon 2% isoflurane. A left lateral incision through the fourth intercostalspace was made to expose the heart. After visualizing the left coronaryartery, a 7-0 or 8-0 prolene suture was placed through the myocardium inthe anterolateral wall and secured as previously described (Kumar, D.,et al., Distinct mouse coronary anatomy and myocardial infarctionconsequent to ligation. Coron Artery Dis, 2005. 16(1): p. 41-4; Singla,D. K., et al., Transplantation of embryonic stem cells into theinfarcted mouse heart: formation of multiple cell types. J Mol CellCardiol, 2006. 40(1): p. 195-200). Coronary artery entrapment wasconfirmed by observing blanching of the distal circulation (ventricularapex). The lungs were over inflated and the ribs and muscle layers wereclosed by absorbable sutures. The skin was closed by additional suturingusing 6-0 nylon or silk.

Epicardial Patch Placement.

Following induction of isoflurane anesthesia (3%), the mouse wasintubated with an 18 gauge catheter and placed on a mouse ventilator at120-130 breaths per minute with a stroke volume of 150 μl and maintainedon 2% isoflurane. A left lateral incision through the fourth intercostalspace was made to expose the heart. The heart was visualized and thesurface dried with a surgical sponge. The seeded patch was placeddirectly onto the epicardial surface of the heart with forceps. Thepatch was then allowed to adhere for 15 minutes before closing thechest. Patch adhesion was subjectively judged with a “sticky factor” bya blinded surgeon who has experience placing epicardial patches. The“sticky factor” ranges from 0-5; 0 being not sticking at all and 5 beingcompletely adhered. After determining the “sticky factor”, the lungswere over inflated and the ribs and muscle layers were closed byabsorbable sutures. The skin was closed by additional suturing using 6-0nylon or silk.

Approximately 48 hours after patch placement, mice were sacrificed byinducing deep anesthesia (3% isoflurane) followed by rapid excision ofthe heart. Hearts were inspected for evidence of patch adhesion thenfixed in 3.6% paraformaldehyde for histological analysis.

hMSC Quantification by CD73 Staining.

Slides containing 5 μm heart sections were probed with goat anti-CD73(Santa Cruz Biotech, Santa Cruz, Calif.) diluted 1:25 in diluting buffer(5% BSA, 0.02% NaN3 in phosphate buffered saline) and incubatedovernight at 4° C. Fluorescence was detected with donkey anti-goat AlexaFluor (AF488, Invitrogen) secondary antibody at 1:200 dilution inpreadsorption solution (90% diluting buffer, 5% human serum and 5% mouseserum) for 45 minutes at room temperature. Samples were counter stainedand mounted with a DAPCO/DAPI solution (2.5% DABCO, 50% glycerol, and0.005% DAPI in PBS). Fluorescence emission was detected on a IX71inverted deconvolution fluorescence microscope (Olympus). Images wereacquired with 20× UPlanFluor objective (NA=0.5), coupled with 3.2×magnification (attaining 64× combined magnification). Analysis wasperformed using Slidebook software (Intelligent Imaging InnovationsDenver, Colo.) and with ImageJ (Fiji, open source software). Images werenormalized to a secondary antibody control. Positive events werecalculated as a percentage CD73 positive divided by total number ofnuclei obtained from analysis of at least eight optical fields persample.

Results

Myocardial Infarction (MI).

We expected to experience some mortality of the mice undergoingexperimental MI. To ensure an adequate number of animals for the patchplacement experiment, MI was induced in twelve animals. Of the twelveanimals, two did not survive the procedure.

Placement of PAA CF 3D-ECM and CF Patches.

Twenty-four hours after induction of MI, the ten surviving miceunderwent placement of patches seeded with 5×10⁵ hMSC; four micereceived CF 3D-ECM patches, and four mice received cardiac fibroblast(CF) patches. The remaining two mice served as non-patch controls. Ofthe mice receiving patches, two did not survive, both from the CF patchgroup. One mouse did not recover from the patch placement surgery andthe second was found dead 48 hours later. Due to this mortality, fourmice made up the CF 3D-ECM patch group, two mice made up the CF patchgroup and two mice were left as non-patch controls. FIG. 8 demonstratesrepresentative examples of CF 3D-ECM and CF patches placed onto theinfarcted myocardium.

Assessment of Patch “Sticky Factor.”

One drawback to many of the materials investigated as potentialepicardial patches is the inability of the patch to adhere or “stick” tothe heart. Because CF 3D-ECM is thin, flexible and uniquely cardiac, wehypothesized that it would more readily adhere to the surface of theheart and not require either sutures or fibrin glue to adhere. Theassessment of a “sticky factor” (scale=0-5, 0=does not stick at all,5=completely stuck down) by the impartial, blinded surgeon, was doneafter the patch had been placed on the surface of the heart for 15minutes. All four CF 3D-ECM were rated as a sticky factor of 4, all fourCF patches were rated as a 3.7. The surgeon noted that the CF 3D-ECMadhered rapidly to the epicardium while CF patches did not, but after 15minutes the patches appeared to be similarly adhered to the epicardium.

Assessment of CF 3D-ECM and CF Patches 48 Hours after Placement.

Forty-eight hours after patch placement, mice were sacrificed and heartsexamined for evidence of patches. Patches remained at the same site ofplacement on the epicardial surface of the heart in all animals (FIG.9). CF patches consistently appeared thicker than CF 3D-ECM patches, asshow in FIG. 9.

Histological Examination of Patches.

Upon removal, hearts were fixed in 3.6% paraformaldehyde, embedded inparaffin, sectioned, and slides stained with hematoxylin and eosin(H&E). Patches were found in all non-control hearts (FIG. 10 A-F).

To further evaluate the adhesion of the patch to the epicardial surfaceof the heart and transfer of hMSCs, immunofluorescence staining wascarried out (FIG. 11). To detect the patch, fibronectin was used, whileCD146, a MSC marker that is not expressed on fibroblasts, was used tovisualize hMSCs. Fibronectin staining was detected, while CD146 wasambiguous. This was due to finding a population of CD146 cells in thecontrol hearts as well. CD146 is also found on epithelial cells as wellas some immune cells. We could not differentiate between transplantedhMSCs and host cells.

To quantify the number of hMSC transferred to the infarcted myocardium,we stained for the hMSC marker CD73 (FIG. 22). The patch and underlyingmyocardium were evaluated for CD73 expression in all eight animals. Inthe CF 3D-ECM patch group, no CD73 expression was found in two out ofthe four animals. In the remaining two animals 3.2% and 2.4% of cells inthe CF 3D-ECM were positive for CD73. No CD73 positive cells weredetected in the myocardium.

In the CF-patch animals 2.9% and 1.4% of the cells in the patch werepositive for CD73. No CD73 positive cells were detected in themyocardium.

Discussion

Determining if CF 3D-ECM Patches Will Adhere to the Heart.

Epicardial patch transfer is a promising method of delivering cells tothe infarcted myocardium. Of the many synthetic and decellularizedtissue patches available, few actually adhere to epicardial surfacewithout the use of sutures or fibrin glue. We accomplished this goal bydemonstrating that both CF 3D-ECM patches and CF patches adhere to theepicardial surface and are located at their placement site after 48hours. Adhesion of the patch to the heart is important for facilitatingtransfer of cells between the patch and the injured myocardium. As isevident in some of the images, when a piece of patch does not maintainadhesion to the heart, the patch retains a high number of cells(nuclei), presumably the seeded hMSC.

Determining if CF 3D-ECM Patches Will Transfer hMSC to the InfarctedMyocardium.

We were able to find a small percentage of cells in the patch that didexpress CD73, but no cells were found in the myocardium. There areseveral possible explanation for this finding. For example, wedemonstrated in Example 1 that even a short time in culture on CF 3D-ECMwill result in large changes in the gene expression profile of BMSC.Therefore it may be reasonable to expect that the hMSC could havedifferentiated or lost their CD73 expression. This would not beunexpected since CD73 is stem cell marker and is lost withdifferentiation. As illustrated in Example 4 below, when using analternative method of detection (FISH), seeded cells did transfer to themyocardium.

Summary.

In this Example, we demonstrated that CF 3D-ECM patches could besuccessfully transferred and adhered to the epicardial surface of aninfarcted heart. Adhesion of the CF 3D-ECM patch did not result indetectable CD73 expression; however, Example 4 below demonstratessuccessful transfer of seeded hMSC cells into the myocardium.

Example 4: Cardiac Fibroblast Patches: Successful Transfer of SeededCells into Target Myocardium

In this Example, we extend our work in the previous examples to (1)further characterize the protein composition of patches, (2) furtherdemonstrate that the patches can be transferred to and maintained on theventricle, and (3) demonstrate that human mesenchymal stromal cells(hMSCs) can be delivered to healthy and infarcted myocardium using thedisclosed ECM patches.

In sum, CFs passage 1-7 from rats were cultured at high density(˜1.6×10⁵/cm²) for 10-14 days during which period the cells secretedlarge amount of extracellular matrix (ECM) to form a manipulablestructure that could be removed from the culture dish followingincubation with EDTA and peracetic acid. Two-dimensional top down massspectrometry demonstrated that the patches were primarily composed offibronectin (81%) with some collagen type I (13%). Additionally, 18non-structural matricellular proteins were identified. Patches could beseeded with hMSC and the seeded patch successfully attached to theepicardial surface of the murine heart without sutures or added glue.Patches remained intact in vivo for at least 2 days. Even at this earlytime point, hMSCs were found to migrate more than 500 μm from theepicardium and infiltrated the myocardium in 8 of 11 mice tested. Insum, this example shows that CF-derived ECM can spontaneously attach toand successfully transfer hMSCs to the infracted heart.

Materials and Methods

Animal Care and Use.

Animals were purchased from Harlan Laboratories. All procedures werecarried out in accordance with protocols approved by the University ofWisconsin School of Medicine and Public Health Animal Care and UseCommittees.

Isolation of Cardiac Fibroblasts.

The technique for isolating cardiac fibroblasts was as performed asdescribed in Example 1. Male Lewis rats (260-400 g) were sacrificed byCO₂ asphyxiation, hearts rapidly excised, atria removed and ventriclesplaced into ice cold PBS with 1% penicillin/streptomycin. Hearts werefinely minced then placed into 10 mL digestion media (Dulbecco'sModified Eagle's Medium (DMEM), 73 U/mL collagenase 2, 2 μg/mLpancreatin (4×)) and incubated at 37° C. with agitation for 35 min. Thedigest mixture was centrifuged at 1000×g for 20 min at 4° C. Theresulting cell pellet was suspended in 10 mL of fresh digestion mediaand incubated at 37° C. with agitation for 30 minutes. The resultingdigest was sieved through a 70 μm cell strainer and digest solutiondiluted with 10 mL of culture media (DMEM, 10% Fetal Bovine Serum (FBS),1% penicillin/streptomycin). The cell suspension was then centrifuged at1000×g for 20 min at 4° C. The cell pellet was suspended in 16 mLculture media and plated into two T75 culture flasks (8 mL per flask).The cells were allowed to attach under standard culture conditions (37°C., 5% CO₂, 100% humidity) for 2 h, then non-adherent cells removed bywashing with PBS and culture media replaced. Primary cardiac fibroblastcultures were typically confluent in 4-7 days.

Generation of CF-ECM. Cardiac fibroblasts, passage 1-7, were plated at adensity of approximately 1.1×10⁵ to 2.2×10⁵ per cm² in high glucoseDMEM+10% FBS and 1% penicillin/streptomycin and cultured at 37° C., 5%CO₂ and 100% humidity for 10 to 14 days. Cardiac fibroblasts andsecreted extracellular matrix were removed from the culture dish byincubation with 2 mM EDTA solution at 37° C. The resulting cardiacfibroblast cell sheet was then denuded of cells by first rinsing twicewith molecular grade water followed by incubation with 0.15% peraceticacid (PAA buffer) for 24-48 hours at 4° C. with constant agitation. Theresulting matrix was then rinsed repeatedly with sterile water followedby PBS.

2D Bottom-Up Mass Spectrometry: This procedure was performed asdescribed in Example 1.

In-Solution Trypsin Digestion.

All solutions were prepared fresh just prior to use with HPLC gradewater. CF-ECM patches were suspended in 15 μL 8M urea and then 20 μL of0.2% ProteaseMax™ added. The CF-ECM was then dissolved into solution byvortexting and pipetting. A volume of 58.5 μL of 50 mM NH₄HCO₃ was addedto a final volume of 93.5 μL. The sample was then reduced by adding 1 μLof 0.5 M DTT and incubating at 56° C. for 20 minutes. 2.7 μL of 0.55 Miodoacetamide was added and incubated for 15 minutes at room temperaturein the dark. 1 μL of 1% ProteaseMax™ and 2 μL of 1 μg/μL Trypsin Gold™were added and incubated overnight at 37° C. The following day 0.5 μL oftrifluorocacetic acid was added to the final concentration of 0.5% tostop the reaction. The sample was then centrifuged at 14,000×g for 10minutes at 4° C. and the cleared supernatant transferred to a fresh 1.5mL protease-free tube.

2D Liquid Chromatography Mass Spectrometry.

2 μL of sample was injected onto an Eksigent 2D nanoLC chromatographysystem and eluted into a Thermo Finnigan LTQ Mass Spectrometer. Thesample was retained on an Agilent Zorbax SB300-C8 trap and eluted byreverse phase gradient onto a 0.100 mm×100 mm emitter packed in-housewith 5 μm bead 300 Å pore MagicC18 material. Mobile phase solutionconsisted of a water and 0.1% formic acid aqueous phase and a 0.1%formic acid in 50% acetonitrile:ethanol organic phase. The gradient ranfrom 1 to 60 min and from 5 to 35% organic with a 95% wash. Eluent wasionized by a positive 3000 V nanoESI and analyzed by a Data Dependenttriple play template. The top 5 m/z were selected by intensity, chargestate was analyzed by zoom scan, and MS/MS were performed with widebandactivation, dynamic exclusion of 1 for 60 s with a list of 300 m/z and awidth of +/−1.5/0.5 m/z, collision energy of 35%, and noise level of3000NL. Sequest searches were performed via Bioworks 3.0 using adownloaded Swissprot database for Rat (October 2010) and its reversedsequences. Search parameters included trypsin digestion, 1 missedcleavage, amino acid length of 6 to 100 with tolerance of 1.4 da,dynamic modifications of methionine methylation (+14 da), and cysteinecarboxyamidomethylation (+57 da). Results were filtered to less than 5%false discovery rate, defined by number of proteins identified withreversed sequences divided by the total number proteins identified minusreversed number, and multiplied by 100.

Confocal Microscopy.

This procedure was performed as described in Example 1. CF-ECM patcheswere fixed in fresh 3.6% paraformaldehyde, embedded in paraffin andsectioned in 5 μm sections and mounted on slides. Slides werede-paraffinized followed by rehydration. Slides were incubated with 0.1%trypsin and a sodium citrate heat retrieval was performed by incubationin 10 mM sodium citrate, 0.05% Tween-20 buffer pH 6 for 60 minutes in anOster® rice steamer (95-100° C.). Slides were blocked with 1% bovineserum albumin in PBST for 1 hour at room temperature then incubated withprimary antibodies (all antibodies purchased from Santa CruzBiotechnology) at a dilution of 1:50 and incubated at 37° C. for 1 hour.Slides were then washed in PBST and incubated in secondary antibodies at1:1000 dilution in 1% bovine serum albumin in PBST (all secondaryantibodies purchased from Invitrogen) for 1 hour at room temperature inthe dark. Slides were washed in PBST and counter stained with 1 μg/mLDAPI for then cover slips mounted with aqueous mounting media and theedges sealed with quick dry, clear nail polish. Slides were imaged atthe W.M Keck Laboratory for Biological Imaging with a Nikon MR scanningconfocal microscope.

Scanning Electron Microscopy.

Sample preparation and imaging were carried out by the Biological andBiomaterials Preparation, Imaging, and Characterization Facility atUniversity of Wisconsin Madison. Briefly, ECM samples were diced with adouble sided razor blade to approximately 3 mm² then fixed in 1%paraformaldehyde, 2% glutaraldehyde in 0.1M sodium cacodylate bufferovernight at 4° C. Samples were washed twice in molecular grade waterthen secondary fixation carried out by incubated with 1% osmiumtetroxide (in water) for 30 minutes. Samples were washed twice inmolecular grade water then dehydrated with a series of 10 minute ethanolincubations (30, 50, 70, 75, 80, 90, 95, and 100%) and sieve dried.Samples were then critical point dried in a Tousimis Samdri 780 fourtimes and ion beam sputter coated with 2.5 nm of platinum. Finally, theprepared samples were imaged on a Hitachi S900 High Resolution FieldEmission Microscope.

Murine Myocardial Infarction.

Myocardial infarction was carried out as previously described (Kumar,D., et al., Distinct mouse coronary anatomy and myocardial infarctionconsequent to ligation. Coron Artery Dis, 2005. 16(1): p. 41-4).Following induction of isoflurane anesthesia (3%), the mouse wasintubated with an 18 gauge catheter and placed on a mouse ventilator at120-130 breaths per minute with a stroke volume of 150 μl and maintainedon 2% isoflurane. A left lateral incision through the fourth intercostalspace was made to expose the heart. After visualizing the left coronaryartery, a 7-0 or 8-0 prolene suture was placed through the myocardium inthe anterolateral wall and secured as previously described (Singla, D.K., et al., Transplantation of embryonic stem cells into the infarctedmouse heart: formation of multiple cell types. J Mol Cell Cardiol, 2006.40(1): p. 195-200). Coronary artery entrapment was confirmed byobserving blanching of the distal circulation (ventricular apex). Thelungs were over inflated and the ribs and muscle layers were closed byabsorbable sutures. The skin was closed by additional suturing using 6-0nylon or silk. A total of 16 mice (5 sham, 11 MI) underwent thoracotomy.Of these, 11 survived the required 48 hours for patch placement andstudy (3 shams, 8 MI).

Human mesenchymal Stromal Cells, Patch Seeding and Placement.

H9 human embryonic stem cell derived mesenchymal stromal cells (hMSC)passage 7 were received as a gift from Dr. Jaehyup Kim and Dr. PeimanHematti of University of Wisconsin-Madison and used as the therapeuticcellular reagent. Twenty four hours post infarction; patches were seededwith hMSCs for 2 hours prior to transfer to the epicardial surface ofthe MI area. The chest was left open for 15 min to allow the patch toseat down prior to closing. Forty-eight hours after the patch wastransferred, mice were sacrificed and hearts examined.

Fluorescence In Situ Hybridization (FISH).

FISH was carried out according to manufacturer's instructions using theTissue Digestion Kit 1 (Kreatech KBI-60007). Briefly, slides werede-paraffinized by baking at 56° C. for 4 hours, followed by xyleneincubation. Slides were rehydrated followed by incubation in 96-98° C.Pretreatment Solution A, then rinsed in water and digested with 200 μlPepsin Solution for 50 minutes at room temperature. Digestion wasstopped by rinsing in water and incubating in 2×SSC buffer at room temp.Slides were dehydrated, then 10 μL All Human Centromere Probe (KreatechKI-20000R) applied to the sample, sealed with a cover slip and incubatedat 80° C. for 5 minutes. Slides were then incubated overnight at 37° C.Slides were washed in Wash buffer II and the cover slip removed thenwashed in Wash buffer I at 72° C. Finally, the slides were washed inWash buffer II, dehydrated and allowed to air dry. Slides were counterstained with DAPI and a cover slip mounted.

Results.

Generation of CF-ECM Patch.

We found that cardiac fibroblasts cultured at high densities for 10-14days could be removed as a cell sheet by incubation with 2 mM EDTA.Duration of culture was dependent primarily on seeding density. Theresulting cardiac fibroblast “sheet” could subsequently bedecellularized to create a cardiac specific extracellular matrix patch,as shown in FIG. 2.

Characterization of CF-ECM Patch.

To characterize the protein composition of the CF-ECM patch,two-dimensional mass spectrometry was performed (n=4). Normalizedspectral abundance factor (NSAF) was used to quantify the abundance ofstructural extracellular matrix proteins (FIG. 13). Fibronectin(82.1+/−2.2%) was found to be the primary component of the matrix withcollagen type I (6.7+/1 0.9% collagen 1A1 and 6.0+/−0.7% collagen 1A2)and collagen type III (3.4+/−0.08%) accounting for a lesser proportionof the matrix. Additionally, small amounts of elastin (1.3+/−0.5%),collagen types II (0.1+/−0.007%), V (0.2+/−0.06%) and XI (0.2+/−0.2%)were detected. Additionally, 18 matricellular proteins were identified(Table 2). Due to the low abundance of matricellular proteins,meaningful quantification was not possible.

TABLE 2 Matricellular proteins Detected in Cardiac ECM MatricellularProteins Transforming growth factor beta 3 Latent transforming growthfactor beta 1 Latent transforming growth factor beta 2 Connective tissuegrowth factor Galectin 1 Galectin 3 Galectin 3 binding protein Matrixmetaloprotease 14 Matrix Gla Protein Granulings SPARC Versican CoreProtein Nidogen 1 VonWillebrand factor A5 A Prothrombin Biglycan Gliaderived nexin Sulfated glycoprotein 1

To evaluate the architecture of the extracellular matrix patch,immunofluorescence microscopy for collagen type I and fibronectin werecarried out. The surface of the decellularized patches were visualizedby fixing and staining (FIG. 14A) while the internal architecture wasvisualized by embedding patches in paraffin and cutting into 5 μmsections (FIG. 14B). Both surface and internal staining corroborated thetwo-dimensional mass spectrometry findings of a matrix rich infibronectin as well as smaller amounts of collagen type I, whichprimarily localized to the surface of the matrix. High resolutionimaging of the surface of the CF-ECM patch using scanning electronmicroscopy revealed a honeycomb like architecture with some cellmembranes and debris present (FIG. 14C).

Attachment of CF-ECM Patch to Epicardium and Transfer of hMSCs into theMurine Myocardium.

To determine if the cardiac extracellular matrix patch could attach tothe epicardial surface without the use of suture or glue, mousemyocardial infarction and sham models were used. A total of 16 mice (5sham, 11 MI) underwent thoracotomy. Twenty four hours post infarction,patches seeded with hMSCs were transferred to the epicardial surface ofthe MI area (FIG. 15A). The chest was left open for 15 minutes to allowthe patch to seat down prior to closing. Eleven mice survived at least48 hours at which point the mice were sacrificed and hearts examined(FIG. 15B). Epicardial patches were adhered to the surface of the heartin 11 of 11 mice. In five of eleven mice, minor adhesion of the patch tothe chest wall or lungs, mostly at the edges of the patch, was observed.Epicardial patch attachment to the heart was confirmed byhematoxylin/eosin (FIG. 15C) and immunofluorescence staining forfibronectin (FIG. 15D).

Evaluation of hMSCs transfer to the infarcted or sham myocardium wascarried out using FISH (FIG. 15). As detailed in Table 2, nucleistaining positive for human centromeres were detected in the adheredextracellular matrix patch in 11 of 11 animals (FIG. 16A). Infiltrationof hMSCs into the myocardium was detected in 8 out of 11 animals withcells primarily residing within 500 μm of the epicardial surface (FIG.16B). In 2 out of 11 samples, hMSCs were detected greater than 500 μmfrom the epicardial surface. In one sample, hMSCs were localized to theendocardial surface of the heart (FIG. 16C).

Discussion

The initial goal of our study was to determine if cell culture anddecellularization techniques could be employed to generate an ECM patchfrom cardiac fibroblasts that harbored structural, mechanical andbiochemical properties necessary for a potential cell-transfer platform.Once this goal was accomplished we proceeded to define the biochemicaland histological properties of the patch. Finally, we determined two keyphysiological features of the patches in vivo: 1) attachment toepicardium, and 2) the ability to bind and release hMSCs thereby actingas a platform for cell transfer.

The CF-ECM patch presented in this example has several notable featureswhich merit discussion; specifically, its physical characteristics andits biochemical composition. We found that physical characteristics ofthe patch such as size, shape and thickness could be manipulated byvarying the size (up to a T75 culture flask) and/or shape of the culturevessel, as well as plating density and length of time in culture (datanot shown). These features are especially important should thistechnology be used in larger animal models and human applications. It isalso important to note that the CF-ECM patch is robust enough to allowfor physical manipulation during the transfer process. Additionally, thedecellularization process developed for the production of CF-ECM patchesdid not employ chemical crosslinking. This may have importantimplication in the immune response and healing process. For example,recent studies show that ECM patches that are not chemically crosslinkedpromote the infiltration and activation of anti-inflammatory macrophages(M2) (see, e.g., Freytes, D. O., L. Santambrogio, and G.Vunjak-Novakovic, Optimizing dynamic interactions between a cardiacpatch and inflammatory host cells. Cells Tissues Organs, 2012. 195(1-2):p. 171-82). These cells have been shown to be associated withimmune-regulatory, remodeling, matrix deposition, and graft acceptance.

To better understand the biochemical nature of the CF-ECM, we usedtwo-dimensional mass spectrometry. To our knowledge, this is the firstrigorous characterization of a fibroblast derived matrix. To date,others studying various fibroblast derived ECM have used antibody basedmicroscopy techniques instead of a discovery tool such as massspectrometry to investigate the composition of the ECM. The CF-ECM wasfound to be predominately fibronectin with lesser amounts of collagensand elastin.

Interestingly, the proportions of ECM proteins found in CF-ECM issimilar, but not identical, to that of a healing myocardium followinginfarction; bearing similarity to the “second order” (fibronectin) scar,which occurs approximately 14 days post infarction (Dobaczewski, M., etal., Extracellular matrix remodeling in canine and mouse myocardialinfarcts. Cell Tissue Res, 2006. 324(3): p. 475-88). These findings mayindicate that CF-ECM in vitro may recapitulates aspects of in vivocardiac healing and thus be a useful new tool for studying the cardiachealing process.

Finally, in regards to biochemical properties of CF-ECM, it is importantto note that we were able to reproducibly detect 18 matricellularproteins using two-dimensional mass spectrometry, as shown in Table 2.These bioactive molecules include growth factors and cytokines areinvolved in important cellular properties such as adhesion, de-adhesion,proliferation, and differentiation.

Implantable biomaterials in the form of epicardial patches maysignificantly increase cellular retention by creating a platform fromwhich therapeutic cells can infiltrate the damaged myocardium. Patchattachment to the epicardial surface is an integral component of thecell transfer strategy. Inherent adhesiveness of the patch is a propertynot currently found in most patches, thus the use of suture or glue toaffix the patch to the surface of the heart is common. This strategy hasthe risk of increasing myocardial damage by restricting small vessels,while tissue glues may not only cause irritation to the surface of theheart but may also inhibit cell migration by acting as a barrier to bothcells and chemokine signals. We found the CF-ECM patches to be adherentin both sham and MI models after an estimated 1.5 to 2 million heartbeats.

Although we did not test the specific nature of the attachment, wespeculate that fibronectin plays a key role due to its expression ofmultiple binding sites for collagen, fibrin/fibronectin, vitronectin,heparin, and cells (through a variety of cell surface integrins). Thehigh fibronectin content of the CF-ECM patch may have additionalbenefits in the healing myocardium as it has been shown to induce cellproliferation, adhesion, survival, and angiogenesis (see, e.g., Berger,S., et al., Short-term fibronectin treatment induces endothelial-likeand angiogenic properties in monocyte-derived immature dendritic cells:Involvement of intracellular VEGF and MAPK regulation. Eur J Cell Biol,2012).

It should be recognized that the composition of the CF-ECM depends onculture conditions. This raises the possibility that the composition ofthe CF-ECM could be experimentally controlled for different clinicalapplication. For example, the composition of a patch intended fortreatment or preventing of cardiac dilation or an aneurysm may require astronger patch perhaps containing more collagen type I.

In summary, we found that the CF-ECM described in this example not onlyadhered to the epicardial surface of the heart, but allowed hMSCs totransfer into the myocardium in 9 out of 11 animals tested. Thisindicates that the hMSCs could reversibly bind to the CF-ECM. Transferof cells was not limited to the epicardial surface immediately underthat patch, but instead hMSCs consistently migrated within the first 500μm of the myocardium. This cellular migration occurred during arelatively brief (48 hour) period and does not appear to be dependent onan “injury signal” since sham as well as MI hearts demonstratedmigration. In both an MI and sham animal, hMSCs had migrated to near theendocardial surface.

Example 5: Increased Plating Density to Make Cardiac Fibroblast3-Dimensional Extracellular Matrix

In this Example, we report the results of increasing the plating densityof the expanded cardiac fibroblast culture that secretes the disclosed3-dimensional cardiac extracellular matrix. We performed the proceduresoutlined in the previous examples, except that we used higher platingdensities than reported in the previous examples. We investigatedplating densities of up to 500,000 cells per cm². Our resultsdemonstrate that these higher plating densities worked just as well asthe previously reported plating densities. However, at plating densitiesbelow 100,000 cells per cm², the 3-dimensional cardiac extracellularmatrix patches do not reliably form.

All references listed in this application are incorporated by referencefor all purposes. While specific embodiments and examples of thedisclosed subject matter have been discussed herein, these examples areillustrative and not restrictive. Many variations will become apparentto those skilled in the art upon review of this specification and theclaims below.

We claim:
 1. A method for preparing a 3-dimensional cardiacextracellular matrix, comprising: (a) isolating cardiac fibroblasts fromcardiac tissue; (b) expanding the cardiac fibroblasts in culture for 1-7passages; and (c) plating the expanded cardiac fibroblasts into aculture having a cell density of 100,000 to 500,000 cells per cm²,wherein the cardiac fibroblasts secrete a 3-dimensional cardiacextracellular matrix having a thickness of 20-500 μm that is attached tothe surface on which the expanded cardiac fibroblasts are plated.
 2. Themethod of claim 1, wherein the cardiac extracellular matrix has athickness of 30-200 μm.
 3. The method of claim 2, wherein the cardiacextracellular matrix has a thickness of 50-150 μm.
 4. The method ofclaim 1, further comprising the steps of contacting the secreted cardiacextracellular matrix with ethylenediaminetetraaceticacid (EDTA), wherebythe cardiac extracellular matrix becomes detached from the surface,forming a free floating bioscaffold.
 5. The method of claim 1, furthercomprising the step of contacting the cardiac extracellular matrix witha decellularizing agent, whereby the cardiac extracellular matrix isdecellularized.
 6. The method of claim 5, wherein the decellularizingagent comprises peracetic acid or a mixture comprising ammoniumhydroxide and octylphenol ethylene oxide (Triton™ X-100).
 7. The methodof claim 1, further comprising the step of seeding the cardiacextracellular matrix with one or more cells that are therapeutic forcardiac disease or injury.
 8. The method of claim 7, wherein the or morecells that are therapeutic for cardiac disease or injury are selectedfrom the group consisting of skeletal myoblasts, embryonic stem cells(ES), induced pluripotent stem cells (iPS), multipotent adult germlinestem cells (maGCSs), bone marrow Mesenchymal stem cells (BMSCs), verysmall embryonic-like stem cells (VSEL cells), endothelial progenitorcells (EPCs), cardiopoietic cells (CPCs), cardiosphere-derived cells(CDCs), multipotent Is/1+ cardiovascular progenitor cells (MICPs),epicardium-derived progenitor cells (EPDCs), adipose-derived stem cells,human mesochymal stem cells, human mesenchymal stem cells (derived fromiPS or ES cells), skeletal myoblasts, or combinations thereof.